key: cord-0051600-263kvo5e
authors: Zhou, Liuying; Liu, Ruijie; Liang, Xujun; Zhang, Sai; Bi, Wu; Yang, Mei; He, Yi; Jin, Jin; Li, Shisheng; Yang, Xinming; Fu, Junjiang; Zhang, Pengfei
title: Long noncoding RNA RP11-624L4.1 is associated with unfavorable prognosis and promotes proliferation through CDK4/6-CyclinD1-Rb-E2F1 pathway in nasopharyngeal carcinoma
date: 2020-10-15
journal: Mol Ther Nucleic Acids
DOI: 10.1016/j.omtn.2020.10.017
sha: 612f3f81c5e0665a5b89870a2658f863967416b7
doc_id: 51600
cord_uid: 263kvo5e

Nasopharyngeal carcinoma (NPC) is one of the most common malignant tumors in southern China and southeast Asia. Emerging evidence revealed that long noncoding RNAs (lncRNAs) might play important roles in the development and progression of many cancers, including NPC. The functions and mechanisms of the vast majority of lncRNAs involved in NPC remain unknown. In this study, a novel lncRNA RP11-624L4.1 was identified in NPC tissues using next-generation sequencing. In situ hybridization (ISH) was used to analyze the correlation between RP11-624L4.1 expression and the clinicopathological features or prognosis in NPC patients. RPISeq predictions and RIP assays were used to identify RP11-624L4.1’s interactions with CDK4. As a result, we found RP11-624L4.1 is hyper-expressed in NPC tissues, which was associated with unfavorable prognosis and clinicopathological features in NPC. By knocking down and over-expressing RP11-624L4.1, we also found that it promotes the proliferation ability of NPC in vitro and in vivo through CDK4/6-CyclinD1-Rb-E2F1 pathway. Overexpression of CDK4 in knocking down RP11-624L4.1 cells can partially rescue NPC promotion, indicating its role of RP11-624L4.1-CDK4/6-CyclinD1–Rb-E2F1 pathway. Taken together, RP11-624L4.1 is required for NPC unfavorable prognosis and proliferation through CDK4/6-CyclinD1-Rb-E2F1 pathway, which may be a novel target for therapeutic and prognostic in patients with NPC.

To investigate the biological functions of RP11-624L4.1 in the development of NPC, 151 we subjected NPC cells to siRNA transfection and shRNA lentiviral infection. Before doing 152 so, we first detected the endogenous expression of RP11-624L4.1 in 6 NPC cell lines ( Figure  153 3A), and we found that RP11-624L4.1 was universally highly expressed in the NPC cell lines 154 compared to NP69 cells (normal human NPE cells). We then selected two cell lines that 155 highly expressed RP11-624L4. 1 showed that RP11-624L4.1 is successfully overexpressed ( Figure 4A) . A CCK8 assay mechanisms by which RP11-624L4.1 exerts its effects on NPC cells proliferation and cell 199 cycle. RPISeq predictions were first used to identify RP11-624L4. 1 

revealed that RP11-624l4.1 may directly interact with CDK4, CDK6 and CyclinD1 ( Figure  201 S5-S7, with probability 0.65, 0.6 and 0.65 respectively using Random Forest classifier, 202 with probability 0.98, 0.94 and 0.89 respectively using SVM). Since CDK4 have the 203 highest probability of binding to RP11-624L4.1, following this indication, RIP assay was 204 performed, and we successfully verified that the RP11-624L4.1 interacts with CDK4 in both 205 5-8F and CNE1 cells ( Figure 6A ). Confocal microscopy of RP11-624L4. 1 FISH and CDK4 206 immunofluorescence showed that they co-localize at the cytoplasm of nasopharyngeal 207 carcinoma cell line 5-8F ( Figure 6D ). 208

for proliferation in NPC in vitro 210 Moreover, we knocked down RP11-624L4.1 expression to investigate whether doing so 211 would alter the mRNA or protein levels of CDK4. As demonstrated by the results, no 212 significant changes were observed in the mRNA levels of CDK4 ( Figure 6B ), but a 213 significant decrease in the protein levels of CDK4 was observed in both the CNE1 and 5-8F 214 cells with a stable knockdown of RP11-624L4.1 ( Figure 6C ). Interestingly, we found that the 215 knocked down of RP11-624L4.1 expression also affects protein expressions/levels for 216 CDK6, CyclinD1, p-Rb (Ser 780), Rb, and E2F1 ( Figure 6C ). Since CDK4 is a 217 protein-serine kinase involved in the cell cycle, we performed a CDK4 218 immunohistochemistry assay and results showed that the relative expression of CDK4 in the 219 sh-1 group was significantly lower than that in the tumors ( Figure 5D&F ). These results 220 indicated that RP11-624L4. 1 showed that overexpression of CDK4 promotes cell proliferation ( Figure 7A ), and partially 229 rescues RP11-624L4.1 nasopharyngeal carcinoma promotion, thus indicating its role in 230 RP11-624L4.1-CDK4/6-CyclinD1-Rb-E2F1 pathway. As we predicted, protein expressions 231 for CyclinD1, p-Rb (Ser 780) and E2F1 were upregulated, whereas Rb was downregulated 232 ( Figure 7B ). Surprisingly, the expression level of CDK6 was slightly reduced. Mostly likely 233 due to RP11-624L4.1 acting as a scaffold of CDK4, CDK6 and CyclinD to form a protein 234 complex, which leads to disruption of distinct regions of this lncRNA to affect different 235 effector partners and function. These results clearly indicated that RP11-624L4.1 promoting 236

In the recent years, increasing numbers of lncRNAs have been proved to play an 245 important role in cancers. In this present study, we showed that RP11-624L4.1 is 246 significantly upregulated in NPC tissues, which had not been previously reported. We 247 determined that a high expression level of RP11-624L4. There are few studies on the mechanism of lncRNA control of CDK4. One 281 mechanism is that lncRNAs can act as decoys to attenuate small RNA regulation through 282 sequestration of proteins or RNA-dependent effectors. The competing endogeneous 283 RNA(ceRNA) hypothesis is based on this hypothesis. 28 and enhances polyubiquitination and degradation of CDK4. 58 We propose that highly 324 expressed RP11-624L4.1 might increase CDK4 expression level through competitive 325 binding SKP2 to inhibit ubiquitin-dependent degradation of CDK4. The specific 326 mechanism needs to be further validated in future studies. 327

We found that RP11-624L4.1 is hyper-expressed in NPC tissues and predicts a poor To measure cell cycle, CNE1 and 5-8F cells were cultured in 6-well plates for 48 hours, 70%~80% of ice-cold ethanol was added, incubated at 4°C for overnight, and washed twice 465 with PBS. PI/RNase staining buffer (BD, USA) was added and incubated at room 466 temperature for 15 minutes in the absence of light. Results were measured by an FACS 467

Calibur system (Cytek NL3000) and analyzed using FlowJo software. All assays were 468 performed in triplicates and representative data were provided. 469

To measure apoptosis, CNE1 and 5-8F cells were stained using the Annexin V kit 470 according to the manufacturer's instructions (BD Biosciences, San Jose, CA, USA). Cells 471 were harvested by trypsinization. Cells were washed once with PBS and then 1×10 6 -10 7 cells 472 were resuspended in 100 µl of 1× binding buffer. FITC-labelled annexin V and propidium 473 iodide (PI) were added to samples and incubated in the dark for 15 min at room temperature 474 (18 to 22°C). Subsequently, cells were subjected to Cytek NL3000 FACS Calibur system. 475

The results were analyzed using FlowJo software. All assays were performed in triplicates.

Next, the tumors were embedded in paraffin and cut into 4.0-µm sections for H&E 489 staining and IHC. H&E staining and IHC were performed according to described protocols. 60, 490 61 Anti-Ki-67 antibody (Beijing Zsbio, China) and anti-CDK4 antibody (Proteintech, China) 491 were used for IHC assay. Images were captured using a Leica DMI3000B inverted 492 microscope (Germany). 493

RIP assays were performed using the Magna RIP RNA-binding protein 495 immunoprecipitation kit (Millipore, USA). Anti-CDK4 antibody (Proteintech, China) and 496 rabbit IgG purified (Millipore, USA) were used for RIP assay. 62 Briefly, 2×10 7 cells were 497 lysed in 0.1 ml of complete RIP lysis buffer containing RIP lysis buffer with protease 498 inhibitors and RNase Inhibitor, and then centrifuged at 14,000 rpm for 10 minutes at 4 °C. 499

The supernatants and magnetic beads were incubated with 5 µg of CDK4 antibody or 500 negative control rabbit IgG at 4 °C overnight with gentle rotation. The beads were washed six 501 times with ice-cold RIP wash buffer. Then, the RNA was purified, and qRT-PCR was 502 performed as described above. 503

Western blotting 504 detection reagent (Beyo ECL Plus, China). The band intensity was quantified by 514 densitometry using ImageJ software (http://rsb.info.nih.gov/ij). GAPDH or α-Tubulin served 515 as internal controls. The experiment was performed three times. 516

Immunofluorescence and RNA FISH 517

For immunofluorescence (IF), cells were fixed on coverslips and permeabilized with 518 4% paraformaldehyde in PBS at room temperature for 10 min, and blocked with 5% normal 519 goat serum in PBS at room temperature for 10 min. Cells were then incubated with primary 520 antibodies at room temperature for 1 h, followed by incubating with secondary antibodies at 521 

The 599 evolution of lncRNA repertoires and expression patterns in tetrapods

Chapter 3: Non-coding RNAs in Cancer

Emerging functional and mechanistic 603 paradigms of mammalian long non-coding RNAs

Long noncoding RNAs: fresh perspectives into the 605 RNA world

The functional role of long non-coding RNA in 607 human carcinomas

Long non-coding RNAs: a new frontier in 609 the study of human diseases

Noncoding RNAs in 611 prostate cancer: the long and the short of it

Cancer genome 613 scanning in plasma: detection of tumor-associated copy number aberrations, single-nucleotide 614 variants, and tumoral heterogeneity by massively parallel sequencing

Noncoding RNA MALAT1 Promotes Aggressive Renal Cell Carcinoma through Ezh2 and 617 Interacts with miR-205

A long noncoding 619 RNA activated by TGF-beta promotes the invasion-metastasis cascade in hepatocellular 620 carcinoma

Targeting Long Noncoding RNA HMMR-AS1 Suppresses and 31

Identification and properties of an atypical catalytic subunit (p34PSK-J3/cdk4) for mammalian D 634 type G1 cyclins

Targeting CDK4 and CDK6: From Discovery to 636 Therapy

Living with or without cyclins and cyclin-dependent 638 kinases

Requirement for CDK4 kinase function in breast cancer

Cdk4 642 disruption renders primary mouse cells resistant to oncogenic transformation

Arf/p53-independent senescence

Evidence 645 for distinct pathomechanisms in B-cell chronic lymphocytic leukemia and mantle cell lymphoma 646 by quantitative expression analysis of cell cycle and apoptosis-associated genes

Clinical and biological significance of CDK4 amplification in well-differentiated and 650 dedifferentiated liposarcomas

cancer cells

PD-0332991, a CDK4/6 inhibitor, significantly prolongs survival in a genetically engineered 656 mouse model of brainstem glioma

Analysis of 44

Long Non-Coding RNA BANCR

Promotes Endometrial Cancer Cell Proliferation and Invasion by Regulating MMP2 and MMP1 672 via ERK/MAPK Signaling Pathway

The LINC01138 drives 674 malignancies via activating arginine methyltransferase 5 in hepatocellular carcinoma

LncRNA-MEG3 inhibits cell 677 proliferation of endometrial carcinoma by repressing Notch signaling

Induced JunD in 680 intestinal epithelial cells represses CDK4 transcription through its proximal promoter region 681 following polyamine depletion

The cell-cycle regulator CDK4: an emerging 683 therapeutic target in melanoma

Cyclin-dependent kinase pathways as targets for cancer treatment

miR-124 downregulation 687 leads to breast cancer progression via LncRNA-MALAT1 regulation and CDK4/E2F1 signal 688 activation

Lnc HAGLR Promotes

Colon Cancer Progression Through Sponging miR-185-5p and Activating CDK4 and CDK6 in 691 vitro and in vivo

LncRNA PCAT-1 693 plays an oncogenic role in epithelial ovarian cancer by modulating cyclinD1/CDK4 expression

Molecular mechanisms of long noncoding RNAs

The steady-state level 704 of CDK4 protein is regulated by antagonistic actions between PAQR4 and SKP2 and involved in 705 tumorigenesis

Drug-resistance Non-small Cell Lung Cancer Progression by Activating AMPK Signaling 708 Pathway

The TWIST/Mi2/NuRD protein 710 complex and its essential role in cancer metastasis

TWIST represses estrogen 712 receptor-alpha expression by recruiting the NuRD protein complex in breast cancer cells

Analysis of lncRNA-Protein Interactions by RNA-Protein Pull-Down

Flotilin-1 promotes 717 the tumorigenicity and progression of malignant phenotype in human lung adenocarcinoma

Tripartite motif 720 containing 28 (TRIM28) promotes breast cancer metastasis by stabilizing TWIST1 protein. Sci

A Brief Review of RNA-Protein Interaction 723 Database Resources. Noncoding RNA. 3

Expressions and 725 significances of the angiotensin-converting enzyme 2 gene, the receptor of SARS-CoV-2 for 726 COVID-19

The expression of RP11-624L4.1 in the gene 733 expression profile (NPE=7; NPC=7). (C) The expression of RP11-624L4.1 in inflammatory 734 NPE tissues (n=14) and NPC tissues (n=20) was determined by RT-qPCR (lower). The 735 results are expressed as log2 (2-∆∆Ct)

Representative ISH staining of RP11-624L4.1, and Kaplan-Meier curve of overall 738 survival (OS) and disease-free survival (DFS) in NPC specimens. (A) Nearly negative 739 expression of RP11-624L4.1; staining intensity level scored as 0. (B) Weak expression of

RP11-624L4.1; staining intensity level scored as 1. (C) Moderate expression of

RP11-624L4.1; staining intensity level scored as 2. (D) Strong expression of RP11-624L4.1; 742 staining intensity level scored as 3; magnification 4×

p-values were calculated 744 with the log-rank test. RP11-624L4.1 highly expressed is associated with an unfavorable 745 prognosis in NPC patients. as determined by CCK-8 cell proliferation assay. (E) A representative photomicrograph and 754 a column chart of EdU assays. Magnification 10×. Scale bar=100 µm. (F) The representative 755 images and quantification column chart of cell cycle assays were measured using the flow 756 cytometry

The 759 overexpression efficiency in the CNE1 and 5-8F cell lines was evaluated by qRT-PCR. OE: 760 cell lines with the overexpression RP11-624L4.1. Vector: cell lines with the transfection with 761 empty-vector DNA. (B-C) The line diagram of the absorbance is determined by CCK-8 cell 762 proliferation assay in OE and vector group. (D) A representative photomicrograph and a 763 column chart of EdU assays. Magnification 10×. Scale bar=100 µm

Knockdown of RP11-624L4.1 suppresses the growth of NPC xenografts

Lentiviral-infected tumor cells were injected into the nude mice, and the volume of the 768 tumors was measured twice per week in the sh-NC and sh-1 groups

Image of the fresh tumors. (C) The final xenograft weights of the sh-NC and sh-1 groups

Data are presented as the mean ± SEM; *p<0.05; Student's t-test. (D) The histomorphology 771 of the tumor sections was analyzed in hematoxylin and eosin (HE)-stained tumor tissues

Rb, E2F1 protein levels. (A) RIP assays of the interaction between RP11-624L4.1 779 and CDK4 by CDK4 antibody in CNE1 and 5-8F cells. The level of RP11-624L4.1 was 780 determined by qRT-PCR and normalized by the input levels. (B) The mRNA expression of 781 CDK4 was determined by RT-qPCR in sh-NC and sh-1 cells. The results are expressed as 782 log2 (2-∆∆Ct)

Rb and E2F1 were detected by western blot (from 3 independent 785 experiments). GAPDH and α-Tubulin were used as loading controls. The band intensity was 786 quantified by ImageJ. (D) Confocal RNA fluorescence ISH and immunofluorescence images 787 are shown the colocalization of CDK4 and

4′,6-diamidino-2-phenylindole

Figure 7. Rescue assay results by overexpressing CDK4 in the knocking down lncRNA

1 cell sh-1. (A) The line diagram of CCK8 cell proliferation assay

Changes in proteins by western blotting in CDK4/CDK6-CyclinD1-Rb-E2F1 pathway

Sh-1+OE, overexpression of GV141-CDK4 in sh-1 which knocked down lncRNA

RP11-624L4.1; Sh-1+vector, without overexpression of CDK4 (GV141 vector only

TNM, tumor-nodes-metastasis, based on the American Joint Committee On Cancer/ 798 International Union Against Cancer Staging Manual