The long non-coding RNA highly expressed in lung cancer was screened by the StarBase V3.0 (http://starbase.sysu.edu.cn/). The miRNA combined with the lncRNA and the possible downstream targets of miR-218-5p were predicted by the most usually used algorithms of TargetScan, miRDB and PicTar, and the overlap part of the three algorithms came into our notice. The putative promoter of miR-218-5p was predicted through Promoter Scan (http://www-bimas.cit.nih.gov/molbio/proscan/).
Cell line and human LUSC tissue
The NCI-H520 cells were cultured with the RPMI-1640 (Gibco, Grand Island, NY, USA) contained 10% fetal bovine serum and 1,000 U/mL P/S. The human LUSC cells were cultured at 37℃ in a humidified atmosphere with 5% CO2. The cell transfection was performed with Lipofectamine 2000 Reagent (Invitrogen, USA) following the manufacturer’s instructions. The NCI-H520 cells and three pairs of human lung squamous cell carcinoma tissues were provided by the Department of Cancer Institute, North China University of Science and Technology Affiliated People’s Hospital. The number, gender, classification and age of the LUSC patients were shown in Table 1. Informed consent was obtained from all subjects or their direct relatives. The cell and tissue studies were submitted to and approved by both the Ethics Committee of North China University of Science and Technology and the Ethics Committee of Hebei Medical University.
Table 1
Clinical Tissue Samples Used in This Work.
Number | Gender | Classification | Age | Date |
370823 | Male | LUSC | 63 | 2019.2.26 |
371406 | Male | LUSC | 50 | 2019.3.6 |
376552 | Male | LUSC | 62 | 2019.4.29 |
qRT-PCR.
The total RNA of lung cancer tissues and cell line were extracted with the mirVana miRNA Isolation Kit (Ambion, USA) following the manufacturer’s instruction. One µg of RNA was reverse transcribed into cDNA with Moloney murine leukemia virus reverse transcriptase (Takara, Japan). The qRT-PCR was conducted with a SYBR® Taq™ kit (Takara Bio, Japan) and the iQ5 Real Time PCR Detection System. The relative fold-change of hsa-miR-218-5p in the transcripts was calculated using U6 as the internal control. The relative fold-change of RNASEH1-AS1, NET1 and POU2F1 were calculated using β-actin as the internal control. All the RT and qPCR primer sequences were listed in the Table 2.
Table 2
The PCR Primers Used in This Work.
Primer Name | Primer Sequence (5’ →3’) |
hsa-miR-218-5p-RT | GTCGTATCCAGTGCAGGGTCCGAGGTGCACTGGATACGACACATGGTTAG |
hsa-miR-218-5p-Fwd | TGCGGTTGTGCTTGATC |
U6 RT | GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACAAAATATGGAAC |
U6 Forward | TGCGGGTGCTCGCTTCGGCAGC |
Reverse | CCAGTGCAGGGTCCGAGGT |
RNASEH1-AS1-qPCR-S | GAGAAGCACCCGCACCTGG |
RNASEH1-AS1-qPCR-AS | GCCTCTAATCCCAACACT |
NET1-qPCR-S | GAAAGGTGAATCCGAGTG |
NET1-qPCR-AS | GTGCCGTTCGTTCCGTGT |
POU2F1-qPCR-S | GTAACCGCCGCCAGAAAG |
POU2F1-qPCR-AS | TGGAGGCTGAGGCAGAAGG |
β-actin-qPCR-S | CGTGACATTAAGGAGAAGCTG |
β-actin-qPCR-AS | CTAGAAGCATTTGCGGTGGAC |
RNASEH1-AS1-Chip-S | TCTGAGGGAGACTGATTC |
RNASEH1-AS1-Chip-AS | ACAGGTGTAGAGTCTGACGTGC |
RNASEH1-AS1-target-Top | AAACTAGGAATTCATGAATTAGAATAAGCACAAAGGAGGGCGAGAGGCT |
RNASEH1-AS1-target-Bot | CTAGAGCCTCTCGCCCTCCTTTGTGCTTATTCTAATTCATGAATTCCTAGTTT |
RNASEH1-AS1-target-mut-Top | AAACTAGGAATTCATGAATTAGAATCGTACTGCAGGAGGGCGAGAGGCT |
RNASEH1-AS1-target-mut-Bot | CTAGAGCCTCTCGCCCTCCTGCAGTACGATTCTAATTCATGAATTCCTAGTTT |
NET1-3’UTR-Top | AAACTAGGAATGATACTATTAAAAAAAAAAAAAGCACACACATAATCACCCTGCT |
NET1-3’UTR-Bot | CTAGAGCAGGGTGATTATGTGTGTGCTTTTTTTTTTTTTAATAGTATCATTCCTAGTTT |
NET1-3’UTR-mut-Top | AAACTAGGAATGATACTATTAAAAAAAAAAACGTACTGCACATAATCACCCTGCT |
NET1-3’UTR-mut-Bot | CTAGAGCAGGGTGATTATGTGCAGTACGTTTTTTTTTTTAATAGTATCATTCCTAGTTT |
POU2F1-3’UTR-Top | AAACTAGGTTGGGGGAAAAAAAAGCACAACTATACCTCTTTAATGTTATTTTCCT |
POU2F1-3’UTR-Bot | CTAGAGGAAAATAACATTAAAGAGGTATAGTTGTGCTTTTTTTTCCCCCAACCTAGTTT |
POU2F1-3’UTR-mut-Top | AAACTAGGTTGGGGGAAAAAACGTACTGCCTATACCTCTTTAATGTTATTTTCCT |
POU2F1-3’UTR-mut-Bot | CTAGAGGAAAATAACATTAAAGAGGTATAGGCAGTACGTTTTTTCCCCCAACCTAGTTT |
sh- POU2F1-Top | GATCCCCAGTCAACACCAAAGCGAATCTCGAGATTCGCTTTGGTGTTGACTGGTTTTTGA |
sh- POU2F1-Bot | AGCTTCAAAAACCAGTCAACACCAAAGCGAATCTCGAGATTCGCTTTGGTGTTGACTGGG |
Sh1- RNASEH1-AS1-top | GATCCGAGAAGCACCCGCACCTGGAGCTCGAGCTCCAGGTGCGGGTGCTTCTCTTTTTGA |
Sh1- RNASEH1-AS1-bot | AGCTTCAAAAAGAGAAGCACCCGCACCTGGAGCTCGAGCTCCAGGTGCGGGTGCTTCTCG |
Plasmid and miRNA mimics.
The miR-218-5p overexpression mimics and ASO-miR-218-5p were commercially synthesized. The sequence was listed in Table 2. The sh-RNASEH1-AS1 and sh-POU2F1 pSilencer vector were generated by annealing the sense and antisense strands of the hairpin RNA following by insertion onto the pSilencer 2.1-U6 neo vector between the BamHI and HindIII sites.
The two strands of both wildtype and mutant 3’-UTR of the NET1 or POU2F1 gene harboring the predicted miR-218-5p binding area were annealed followed by insertion into the upstream of the reporter gene of pmirGLO vector. Similarly, the putative promoter area and miR-218-5p binding region of lnc-RNASEH1-AS1 were amplified by PCR and inserted into the upstream of the reporter gene of pGL3-basic-luciferase vector.
All the insertions mentioned above were verified by DNA sequencing. All primers used in this work were shown in Table 2.
Dual-luciferase reporter assay.
NCI-H520 cells were cultivated in 48 well plate at a density of 6 × 104 every well. miR-218-5p transfection reagents were prepared at the final density of 20 µM and incubated for 4 h. After 24 h, pmirGLO-targetgene-3’UTR /mut transfection regents were added into cultured cells at the final density of 0.5 µg. After transfection for 4 h, the transfection mixture was replaced with 300 µL of new complete 1640 medium. After cotransfection for 48 hours, the luciferase activity was tested following the instructions of the Dual-Luciferase Reporter System (Thermo, USA).
Cell viability and proliferation assay.
In the MTT assay, NCI-H520 (1000 per well) cells were planted into 96 well plates. 24, 48, and 72 h after transfection, MTT was added into every well, and the plates were cultured for 4 h. The absorbance at a wavelength of 570 nm was measured to evaluate the cell viability.
In the colony formation assay, NCI-H520 (200 per well) cells after transfection were trypsinized, and planted into 12 well plates and cultured for 7 to14 days at 37 °C. The colonies were dyed with dyeing solution with 0.2% crystal violet and 20% methanol. Colonies of cell number over 50 were counted and analyzed. Colony formation rate calculating: colony formation rate = (number of colonies / number of seeded cells) × 100%. All cell experiments were conducted for over three times.
Trans-well invasion and wound healing assay.
In the trans-well invasion assay, NCI-H520 cells (1 × 105 per well) were put into the upper chamber of every insert (Milipore, USA) containing 50 µl of matrigel (Milipore, USA). 800 µl of DMEM medium supplemented with 20% FBS (JIBCO, USA) was added onto the lower chambers. After 72 h, cells attached to the lower surface were stained for 15 min with crystal violet. After that we take pictures for counting.
In the wound-healing assay, NCI-H520 cells were cultured in 12 well plates. When cell confluence reached 70%-80%, scratches were made by a 50 ml pipette tip, and non-adherent cells were removed by three times of PBS wash. Wounded cells were cultured in medium containing non FBS for 0, 24, and 48 hour. Three fields of view were randomly captured for every well.
Western blot.
NCI-H520 cells were collected and lysed with lysis buffer (100 mM Tris-HCl, 2% SDS, 1 mM Mercaptoethanol and 25% Glycerol). Cell extracts were heated in loading buffer and the same amount of cell extracts were run on a 10% SDS-PAGE gels. After being electrophoretically transferred to a PVDF membranes (Millipore, USA), the protein bands were probed with its corresponding primary antibodies (anti-NET1, anti-POU2F1, Saier Biotechnology, china), and cultured overnight at 4 °C. The secondary antibody (anti-GAPDH, Saier Biotechnology, Tianjin, China) was added and incubated at room temperature for 1.5 hour. The PVDF membranes were washed in PBS for four times and the immunoreactive target bands were visualized by the chemiluminescence imaging system (Huqiu Image Instruments, Suzhou, China). The band intensities were quantified by Lab Works image analysis software (UVP, USA).
Animal model.
Twenty BALB/c-nu mice (female) of 5 to 6 week old were purchased from the institute of experimental animals, Chinese academy of medical sciences (Beijing). They were randomly divided into two groups. In total, 2 × 107 NCI-H520 cells were transfected with siR- RNASEH1-AS1 or siR-NC and were suspended in 100 µl of serum free RPMI1640 for every nude mouse. The cells suspension was injected directly into the left back of the mice. The tumor volume was measured every three days after injection. Four weeks later, the mice were sacrificed by the method of cervical dislocation after anesthesia, and their tumors mass were harvested. No mice dead before this. The tumor weight was measured and the average tumor weight was calculated. The tumor tissues were stored in -80℃ or used to perform Hematoxylin-Eosin (HE) and immumohistochemical staining. All studies were performed under the American Association for the Accreditation of Laboratory Animal Care guidelines and adhered to national and international standards. All animal works were approved by the Ethics Committee of Hebei Medical University.
CHIP assay.
The combination of POU2F1 to promoter of lnc- RNASEH1-AS1 was confirmed by chip assay following the instructions of Chromatin Immunoprecipitation Kit (milipore, USA). Primers flanking the predicted POU2F1 binding site in the lnc- RNASEH1-AS1 promoter were used in PCR. DNAs were purified using the Chromatin Immunoprecipitation Kit (milipore, USA). The primers used in this study were shown in Table 2.
Statistical evalution.
The data was analyzed by GraphPad Prism 6 Software (GraphPad Software, USA) with the two-tailed Student’s t test. The results were presented as mean ± S.D. of three separate experiments. Unpaired Student’s t-test was used to compare the two groups. p value of less than 0.05 was regarded as statistically significant (* p༜0.05, ** p༜0.01, ***p༜0.001).