LncRNA MIR210HG Facilitates Non-Small Cell Lung Cancer Progression Through Directly Regulation of miR-874/STAT3 Axis.

BACKGROUND: Long noncoding RNAs are involved in the progression of multiple cancers. However, the expression and mechanism of microRNA (miR)210HG in non-small cell lung cancer (NSCLC) remain unclear.
METHODS: The levels of miR210HG and miR-874 were measured by quantitative real-time polymerase chain reaction in NSCLC tissue samples and cells. Non-small cell lung cancer cell proliferation, migration, and invasion were measured by Cell Counting Kit-8 and transwell assays. Luciferase analysis confirmed the interaction between miR210HG and miR-874.
RESULTS: Here, our data showed that miR210HG was overexpressed in NSCLC tissue samples and cells. In vitro functional assays showed that silencing miR210HG blocked NSCLC cell proliferation, migration, and invasion while promoting NSCLC cell radiosensitivity and chemoresistance. Mechanistically, miR-874 was directly regulated by miR210HG. Furthermore, miR-874 expression was reduced in NSCLC tissues and cells. The miR-874 mimic could mitigate the promoting effect of miR210HG on NSCLC cell progression. The data also showed that miR210HG promoted NSCLC cell progression through miR-181a expression by targeting STAT3.
CONCLUSIONS: Our observations suggest that miR210HG is associated with NSCLC cell progression by regulating the miR-874/STAT3 axis.

Introduction

Non-small cell lung cancer (NSCLC) is the subtype with the most lung cancer–related deaths worldwide.[1,2] Although multiple therapeutic strategies have been developed, more detail about NSCLC pathogenesis remains to be revealed.[1,3-5] Therefore, it is urgent to determine new possible mechanisms for NSCLC treatment.

Long noncoding RNAs (lncRNAs) are classified as transcribed RNA sequences >200 nucleotides and lack protein-coding capacity.[6,7] Long noncoding RNA functions as a key regulator in cell growth,[8,9] metastasis,[10] invasion,[11] and differentiation.[12] Moreover, lncRNAs also have key physiological effects on cancer progression.[13-16] Long noncoding RNA DGCR5 induces NSCLC progression by regulating the microRNA (miR)-330-5p/CD44 axis.[17] Long noncoding RNA DUXAP9-206 promotes NSCLC development via the EGFR pathway.[18] Long noncoding RNA LOC285194 is a suppressor of NSCLC progression by targeting p53.[19] Long noncoding RNA MALAT1 reduces NSCLC cell chemosensitivity by regulating the miR-197-3p/p120 catenin pathway.[20] Moreover, the biological mechanism of other lncRNAs in NSCLC progression has not been investigated.

A previous study revealed that lncRNA miR210HG is upregulated in hepatocellular carcinoma tissues compared to control samples.[21] Furthermore, miR210HG might also serve as a potential oncogenic regulator in colorectal adenocarcinoma[22] and glioma.[23] To date, the level of miR210HG in NSCLC remains largely unknown. Additionally, the role of miR210HG in NSCLC metastasis and other processes have not been investigated. In this study, we demonstrated the miR210HG expression pattern in NSCLC tissues and cells and the possible molecular mechanism.

Materials and Methods

Patient Sample Collection

NSCLC patient samples (32) and matched control samples (32) were harvested at First People’s Hospital of Yunnan Province between May 2016 and August 2017. The samples were stored in liquid nitrogen immediately after surgery. This study was approved by the ethics committee of First People’s Hospital of Yunnan Province.

Cell Culture

Four human NSCLC cell lines (NCI-H1975, H1299, A549, and GLC-82) and the normal human bronchial epithelial cell line 16HBE were purchased from ScienCell Research Laboratories (Carlsbad, California) and cultured in RPMI 1640 medium (Gibco, Carlsbad, California) with 10% fetal bovine serum (FBS; Gibco, Grand Island, New York) at 37°C with 5% CO2.

Cell Transfection

The siRNA against miR210HG (si-miR210HG), pcDNA3.1-miR210HG, pcDNA3.1-NC, miR-874 mimics, miR-874 inhibitor, and the negative controls (GenePharma, Shanghai, China) were transfected into NSCLC cells using Lipofectamine 3000 (Invitrogen, Carlsbad, California) based on the manufacturer’s protocol for 48 hours.

RNA Extraction and Quantitative Real-Time PCR

Total RNA from NSCLC samples and cells was harvested by TRIzol reagent (Invitrogen, Carlsbad, California) based on the manufacturer’s protocol. The expression of miR210HG was determined using SYBR Green Master Mixture reagent (MedChemExpress, Shanghai, China). The miR-874 level was analyzed using TaqMan miRNA assays (Thermo Fisher Scientific, Shanghai, China). The expression of miR210HG and miR-874 was normalized against the expression of glyceraldehyde 3-phosphate dehydrogenase and U6, respectively. The 2−ΔΔCt method was used to calculate the relative gene level.

Colony Formation Assay

Non-small cell lung cancer cells were incubated in 6-well plates for 14 days at 37°C, and the colonies were fixed with 4% paraformaldehyde for 10 minutes and dyed with 0.1% crystal violet (Beyotime Institute of Biotechnology, Shanghai, China) for 10 minutes at room temperature.

Cell Proliferation Assay

Cell Counting Kit-8 (CCK-8; Dojindo, Kumamoto, Japan) was used to detect NSCLC cell proliferation as previously described. Non-small cell lung cancer cells (1 × 104) were incubated in 96-well plates for the indicated time periods. Then, pemetrexed, paclitaxel, or carboplatin were added to the plate for 24 hours. Next, 10 µL of CCK-8 solution was added to the plates for 2 hours. The absorbance values were measured at 450 nm.

Radiosensitivity Assay

Non-small cell lung cancer cells (1 × 104 cells/well) were seeded into a 96-well plate. Then, cells were exposed to different doses (0, 4, or 8 Gy) of radiation (IR) for 72 hours. Finally, the radiosensitivity was determined by a CCK-8 assay.

Transwell Migration and Invasion Assays

The upper insert was precoated with Matrigel, and 1 × 105 NSCLC cells were resuspended in 100-µL RPMI 1640 medium without FBS and added to the upper 8-mm pore insert (Corning, Bedford, Massachusetts). Then, 600-µL RPMI 1640 medium plus 20% FBS was added to the lower insert, and the plate was incubated at 37°C for 12 hours. The cells were stained with 0.1% calcein-AM for 15 minutes. Cell migration was evaluated in transwell inserts without Matrigel. The rate of migrated NSCLC cells was analyzed by imaging the cells in randomly selected fields of vision from 3 independent tests followed by quantification with ImageJ software (NIH Image).

Luciferase Reporter Assay

To investigate the relationship between miR210HG and miR-874, NSCLC cells were cotransfected with pGL3-luciferase reporter vectors (OBIO, Shanghai, China) and miR210HG-WT–3’UTR or miR210HG-MUT–3’UTR and miR-NC or miR-874 mimic by Lipofectamine 3000 based on the manufacturer’s instructions for 48 hours. Luciferase activities were analyzed by the dual-luciferase reporter assay (Promega, Madison, Wisconsin).

Western blot Analysis

Hepatocellular carcinoma cells were lysed with RIPA buffer (Beyotime, Beijing, China). The lysates were separated by 10% sodium dodecyl sulphide–polyacrylamide gel electrophoresis and electroblotted onto PVDF membranes. The blots were treated with 5% BSA for 1 hour, probed with primary antibody at 4°C overnight, and then probed with the appropriate secondary antibody for 1 hour at room temperature. The signals were visualized by enhanced chemiluminescence (Pierce, Rockford, Illinois).

Statistical Analysis

Data are expressed as the mean ± standard deviation of 3 independent experiments. All statistical analyses were performed using SPSS version 20.0 software (SPSS, Chicago, Illinois). Differences between groups were evaluated by Student t test or 1-way analysis of variance when more than 2 groups were compared. P < .05 was regarded as statistically significant.

Results

miR210HG Is Upregulated in NSCLC Samples and Cell Lines

The expression pattern of miR210HG was detected in NSCLC tissues and cells. The data revealed that miR210HG was highly expressed in NSCLC tissues (Figure 1A). Next, we examined the expression of miR210HG in NSCLC cells. Similarly, the level of miR210HG was higher in NSCLC cell lines (Figure 1B). Therefore, we hypothesized that miR210HG might be associated with NSCLC progression.

Figure 1.

The expression of miR210HG increases in NSCLC samples and cells. A, the relative expression of miR210HG was detected in NSCLC tissues and paired normal samples. B, The relative expression of miR210HG was detected in NSCLC cells compared with the normal human bronchial epithelial cell line 16HBE. *P <.05, **P < .01. miR210HG indicates micro RNA; NSCLC, non-small cell lung cancer.

miR210HG Contributes to the Proliferation and Chemoresistance of NSCLC Cells In Vitro

To study the roles of miR210HG in NSCLC in depth, loss-of-function experiments were performed. The expression of miR210HG was reduced in NSCLC cells upon si-miR210HG transfection (Figure 2A). The colony formation assay results showed that the clone-forming ability was decreased in NSCLC cells after miR210HG knockdown (Figure 2B). Moreover, CCK-8 data demonstrated that knockdown of miR210HG reduced the proliferation capacity of NSCLC cells (Figure 2C and D). Thus, these results revealed that miR210HG could contribute to the proliferation of NSCLC cells in vitro.

Figure 2.

miR210HG modulates NSCLC cell proliferation. A, After NSCLC cells were transfected with si-miR210HG, the relative expression of miR210HG was determined by qRT-PCR. B, Colony formation assay revealed that miR210HG knockdown repressed the proliferation of NSCLC cells. C and D, Cell Counting Kit-8 assay indicated that miR210HG knockdown inhibited the proliferation of NSCLC cells. E and F, Radiosensitivity assays indicated that miR210HG knockdown induced the radiosensitivity of NSCLC cells. G and H, The CCK-8 assay indicated that miR210HG knockdown induced the chemoresistance of NSCLC cells. *P < .05, **P <.01. qRT-PCR indicates quantitative real-time polymerase chain reaction. miR210HG indicates micro RNA; NSCLC, non-small cell lung cancer.

Next, the effect of miR210HG on NSCLC cells treated with IR. The CCK-8 results showed that knockdown of miR210HG enhanced the radiosensitivity of A549 and H1299 cells (Figure 2E and F).

Furthermore, we found that knockdown of miR210HG resulted in a lower survival rate of A549 and H1299 cells than the NC group, even after treatment with pemetrexed, paclitaxel, or carboplatin (P < .05; Figure 2G and H). These data showed that miR210HG may induce NSCLC radiosensitivity and chemoresistance.

miR210HG Contributes to the Invasion and Migration of NSCLC Cells In Vitro

To study the role of miR210HG in the metastatic potential of NSCLC cells in depth, the migration and invasion abilities were measured. We observed that knockdown of miR210HG could decrease NSCLC cell migration in vitro (Figure 3A and B). Transwell invasion assays showed that silencing miR210HG could repress NSCLC cell invasion (Figure 3C and D). These data indicated that miR210HG could contribute to NSCLC cell invasion and migration in vitro.

Figure 3.

miR210HG modulates NSCLC cell migration and invasion. A and B, transwell analysis revealed that miR210HG knockdown repressed the migration of NSCLC cells. C and D, the matrigel invasion assay indicated that miR210HG knockdown repressed the invasion of NSCLC cells. *P < .05, **P < .01. miR210HG indicates micro RNA; NSCLC, non-small cell lung cancer.

miR-874 Directly Binds to the miR210HG 3′-UTR

To identify potential targets of miR210HG, bioinformatics assays and luciferase reporter assays were performed. The putative binding sites between miR-874 and miR210HG are shown (Figure 4A). To further detect whether miR210HG could target miR-874, a luciferase reporter assay was used. The data showed that the miR-874 mimic reduced the luciferase activity of miR210HG-WT, demonstrating that miR210HG is a direct target of miR-874 (Figure 4B). Next, we also found that miR-874 levels were decreased in NSCLC tissues and cell lines (Figure 4C and D). Collectively, the above experiments revealed that miR-874 could directly bind with the miR210HG 3′-UTR.

Figure 4.

miR-874 directly targets miR210HG. A, miR-874 could interact with the 3’UTR of miR210HG. B, A luciferase reporter assay was performed in NSCLC cells cotransfected with WT-miR210HG or MUT-miR210HG and miR-874. C and D, The relative expression of miR-874 was evaluated by qRT-PCR assay in NSCLC samples and cell lines. *P < .05, **P < .01. qRT-PCR indicates quantitative real-time polymerase chain reaction. miR-874 indicates micro RNA; NSCLC, non-small cell lung cancer.

miR-874 Rescues the Functions of miR210HG in NSCLC Cells

To study whether miR210HG promotes NSCLC cell progression by targeting miR-874 in detail, a rescue assay was performed. The miR-874 mimics inhibited NSCLC cells migration and invasion (Figure 5A and B). As expected, the colony formation assay showed that miR-874 mimics could reverse the promoting effect of miR210HG overexpression on NSCLC cell colony formation (Figure 5C). The metastasis and invasion assay showed that miR-874 mimics could rescue the promoting effect of miR210HG overexpression on NSCLC cell migration and invasion (Figure 5D and E). This evidence indicated that miR-874 could rescue the promoting effect of miR210HG in NSCLC cell progression.

Figure 5.

miR-874 reverses the effect of miR210HG on NSCLC cell progression. A and B, miR-874 mimic repressed the migration and invasion of NSCLC cells. C, miR-874 mimic decreased miR210HG overexpression-induced NSCLC cell proliferation. D and E, miR-874 mimic decreased miR210HG overexpression-induced NSCLC cell migration and invasion. *P < .05, **P < .01. miR-874 indicates micro RNA; NSCLC, non-small cell lung cancer.

STAT3 Regulates the Effects of miR210HG on NSCLC Cells

Next, we detected whether miR210HG modulates the expression of STAT3 via miR-874 in NSCLC cells. The quantitative real-time polymerase chain reaction data showed that miR-874 mimics reduced the expression of STAT3, while the miR-874 inhibitor had the opposite effect (Figure 6A). Moreover, NSCLC cells were transfected with si-miR210HG and miR-874 inhibitor. As shown in Figure 6B, si-miR210HG decreased STAT3 expression, and this effect was abolished by miR-874 inhibitor treatment. Therefore, our data suggest that miR-874/STAT3 may contribute to the effect of si-miR210HG on NSCLC progression.

Figure 6.

miR210HG elevates STAT3 expression in NSCLC cells. A, STAT3 expression was detected in NSCLC cells treated with miR-874 mimic, miR-874 inhibitor, and control. B, The level of STAT3 was detected in NSCLC cells cotransfected with si-miR210HG and miR-874 inhibitor. C-E, cell proliferation, migration and invasion were determined in NSCLC cells cotransfected with si-miR210HG and pcDNA-STAT3. *P < .05, **P < .01. miR210HG indicates micro RNA; NSCLC, non-small cell lung cancer.

To characterize whether the effect of miR210HG on NSCLC development is mediated by STAT3, NSCLC cells were cotransfected with si-miR210HG and pcDNA-STAT3. The data indicated that upregulation of STAT3 partially rescued the effect of si-miR210HG on NSCLC development (Figure 6C–E), suggesting that STAT3 mediates the effects of miR210HG on NSCLC development.

Discussion

The vital role of lncRNAs in the occurrence and development of NSCLC has been discovered and confirmed. Long noncoding RNAs are reported to have many roles in NSCLC progression. Long noncoding RNAs MALAT1 is upregulated in NSCLC tissues. MALAT1 also upregulates NSCLC proliferation and metastasis of NSCLC cells by interacting with the Rac1/JNK signaling pathway. Furthermore, overexpression of MALAT1 is correlated with poor survival in NSCLC patients. CPNE3 facilitates NSCLC migration and invasion via the RACK1/FAK pathway. These studies demonstrate the comprehensive effects of lncRNAs in NSCLC tumorigenesis. However, the expression and role of lncRNA miR210HG in NSCLC remain largely unclear.

Long noncoding RNAs miR210HG is abnormally expressed in many tumors, including hepatocellular carcinoma, colorectal adenocarcinoma, and glioma. Consistent with these previous results, our study reported that miR210HG was significantly upregulated in NSCLC tissue samples and cell lines. Loss-of-function assays demonstrated that miR210HG knockdown inhibited NSCLC cell proliferation in vitro. In addition, miR210HG knockdown inhibited the migration and invasion of cells in vitro and resulted in increased radiosensitivity and chemosensitivity compared with the controls. Thus, our results revealed the suppressive effect of miR210HG knockdown on NSCLC cell invasion and metastasis. Thus, miR210HG could serve as a migratory and invasive regulator in NSCLC cells, which is a key step leading to NSCLC progression.

Some reports have shown that lncRNA miR210HG functions as a miRNA sponge to carry out its key function in hepatocellular carcinoma, colorectal adenocarcinoma, and glioma. Investigating these data, miR210HG has been verified to influence the expression of miR-1226-3p and miR-503 in other types of tumors. Based on the bioinformatics results, we found that miR-874 is a downstream target of miR210HG. The luciferase reporter assay verified that miR-874 directly binds with the miR210HG 3′-UTR. A rescue assay showed that miR-874 could reverse the oncogenic function of miR210HG in NSCLC cells. These data indicated that miR-874 at least partly mediated the oncogenic effect of miR210HG on NSCLC cells.

Previous studies have shown that STAT3 is a target of miR-874 in NSCLC cells. Here, we observed that the levels of STAT3 were negatively affected by miR-874 in NSCLC cells. Furthermore, silencing of miR210HG decreased NSCLC progression, which was reversed by overexpression of STAT3, indicating that miR210HG negatively regulates the expression of miR-874 and thus promotes the expression of STAT3 in NSCLC cells. We also found that the inhibitory effects on NSCLC cell progression induced by miR210HG knockdown were reversed by overexpression of STAT3, suggesting that miR210HG promotes NSCLC progression via regulation of miR-874 and STAT3.

To conclude, this study revealed the upregulation of miR210HG in NSCLC tissues and cell lines. Moreover, the results demonstrated that miR210HG acts as an oncogenic supervisor of NSCLC proliferation, invasion, metastasis, radiosensitivity, and chemoresistance by targeting the miR-874/STAT3 axis.