Effects of N6-methyladenosine-induced LncRNA PVT1 targeting MYC on the stemness of ketamine-treated breast cancer cells / 中华内分泌外科杂志

Objective:To investigate the effect of ketamine on dryness maintenance of breast cancer (BC) cells by regulating LncRNA PVT1/MYC axis.Methods:BC cell line MCF-7 was treated with different concentration of ketamine (0, 5, 10 or 20 g/ml) or treated with 20g/ml ketamine for different periods (0, 24, 48 or 72h) . Furthermore, the expression of METTL3, PVT1 and MYC in MCF-7 cells was interfered and MCF-7 cells were divided into different groups.Western blot was used to detect the expression levels of stem cell characteristic related molecules (OCT4 and SOX2) . The expression level of PVT1/MYC in each group was detected by qRT-PCR. MeRIP analysis was used to detect THE m6A methylation level of PVT1.Results:Ketamine treatment significantly reduced the number of BC globules and inhibited the protein expression of OCT4 and SOX2 in a dose-and time-dependent manner (all P<0.05) . Ketamine regulated m6A level of METTL3-mediated PVT1. Compared with ketamine+pcDNA3.1 group (207±11) , the number of globules formed in ketamine+PVT1 group (311±15) was significantly increased ( t=12.06, P<0.001) , and the protein expression levels of OCT4 and SOX2 were increased ( t=9.68, P<0.001; t=11.50, P<0.001) . MYC was a downstream regulatory gene of PVT1. Compared with ketamine+PVT1+ Si-NC group, ketamine+PVT1+si-MYC group significantly reduced the number of spheroid formation ( t=0.54, P=0.005) and the expression levels of OCT4 and SOX2 proteins ( t=5.98, P=0.004) ( t=7.33, P=0.002) . Conclusion:Ketamine mediates the expression of PVT1 and its downstream gene MYC by inhibiting THE m6A level of PVT1, thus inhibiting the stem cell-like characteristics of BC cells.

Circular RNA PVT1 Promotes Proliferation and Represses Apoptosis of Colorectal Cancer Cells / 中国生物化学与分子生物学报

Circular RNAs (circRNAs) are a novel class of long-chain non-coding RNAs. Preceding evidence has showed that circRNAs participate in the development and progression of various tumors. In the present study, we investigated the expression of circRNAs in 5 paired colorectal cancer (CRC) tissues and adjacent normal tissues with circRNA high-throughput sequencing. Totally 477 differentially expressed circRNAs were identified between CRC tissues and non-cancerous matched tissues, which included 252 significantly overexpressed circRNAs and 225 downregulated circRNAs. CircRNA plasmacytoma variant translocation 1 (circPVT1), the most up-regulated expression circRNA, was further confirmed by qRT-PCR in 150 colorectal cancer tissues and matched normal mucosae. Our data revealed that circPVT1 showed a significant up-regulation trend in CRC tissues compared with matched normal mucosae. Similarly, compared with normal colorectal mucosa cells, the expression of circPVT1 in colorectal cancer cell lines was significantly up-regulated (P<0. 05). Functionally, silence with siRNA or overexpression of circPVT1 in colorectal cancer cells was applied to determine the biological functions of circPVT1, including cell proliferation, apoptosis, and cell cycle, etc. The results show that circPVT1 expression significantly attenuated apoptosis, induced replication and promoted proliferation of colorectal cancer cells in vitro. In summary, our findings indicate that circPVT1 plays an oncogenic role in CRC and might be a potential novel target for CRC therapy.

Expression of lncRNA PVT1 in colorectal cancer tissues and cells and its effect on chemo-sensitivity to cisplatin and the possible mechanisms / 中国肿瘤生物治疗杂志

@#Objective: To study the regulatory effects and possible mechanism of long non-coding RNA plasmacytoma variant translocation 1 (lncRNA PVT1) on chemotherapy sensitivity to cisplatin (DDP) of colorectal cancer (CRC)．Methods: A total of 112 pairs of matched cancer and adjacent non-cancerous tissues were obtained from the CRC patients who underwent surgical resection in the First Affiliated Hospital of Xinxiang Medical University betweenApril 2006 and March 2011.All specimens were confirmed by pathological examinations. Tumor tissues and corresponding adjacent non-cancerous tissues from 30 cisplatin-sensitive CRC patients and 30 cisplatin-resistant patients were selected. Human CRC cell lines (HT29, SW480, HCT116, RKO and LoVo) and normal colonic epithelial cell line NCM460 were also collected for this study; and DDP-resistant RKO/DDP and LoVo/DDP cell lines were constructed. siPVT1, siNC, LV-PVT1 and LV-NC were transfected into LoVo and RKO cells or LoVo/DDP and RKO/DDP cells using lipofectamineTM2000. The expression of lncRNA PVT1 in CRC tissues and cells was tested by Real-time qPCR. CCK-8 assay, flow cytometry and WB were performed to test the effect of PTV1 knockout or enforcement on cell proliferation, apoptosis and expressions of apoptosis-related proteins, respectively. The CRC subcutaneous transplanted xenograft model was established on athymic nude mice to study the effect of PVT1 over-expression on tumor growth and DDP resistance. Results: PVT1 was highly expressed in the cancer tissues and CRC cells, and its expression was positively associated with cisplatin resistance of CRC. After knockdown of PVT1, the proliferation of cisplatinresistant CRC cells was significantly suppressed, while the apoptosis was significantly enhanced (P<0.05 or P<0.01); Mechanically, the levels of drug resistance-associated molecules, including MDR1 and MRP1, as well as the expression of anti-apoptotic Bcl-2 were significantly downregulated whereas the levels of pro-apoptotic Bax and cleaved caspase-3 were increased in PVT1-silenced DDP-resistant CRC cells. Over-expression of PVT1 reversely increased proliferation and decreased apoptosis of CRC cells (P<0.05 or P<0.01). In addition, PVT1 over-expression in CRC cells significantly promoted DDP-resistance in vivo (P<0.05). Conclusion: Collectively, knockdown of PVT1 expression can significantly suppress cell proliferation and promote apoptosis of DDP-resistant CRC cells. Overexpression of PVT1 can significantly promote the growth of CRC cells in vitro and transplanted xenograft in vivo. PVT1 regulates endogenous apoptosis pathways and further promotes the sensitivity of CRC cells to cisplatin chemotherapy via inhibiting the expressions of MDR1 and MRP1.

Effect of lincRNA PVT1 associated with Enhancer of Zeste homolog 2 (EZH2) and with androgen receptor (AR) on the large-scale gene expression in LNCaP prostate cancer cells

O lincRNA PVT1 (Plasmacytoma Variant Translocation 1) é um RNA longo não codificador de proteínas (ncRNA) descrito como um oncogene sendo superexpresso em vários tipos de cânceres. LincRNA PVT1 está localizado na região genômica 8q24, também conhecida como 'gene desert'. O nível de expressão do lincRNA PVT1 está associado ao aumento do risco de câncer de próstata (PCa) e está correlacionado com os níveis de expressão do receptor de andrógeno (AR). No entanto, o mecanismo do envolvimento do lincRNA PVT1 com o AR no desenvolvimento de câncer de próstata ainda não está bem esclarecido. Aqui, nós testamos a hipótese que a formação do complexo AR-EZH2-PVT1 participa na regulação da expressão gênica em câncer de próstata, nas células LNCaP. A imunoprecipitação de ribonucleoproteínas seguida de PCR quantitativo (RIP-qPCR) revelou que o lincRNA PVT1 está associado fisicamente ao AR (12% do input) e à metiltransferase EZH2, proteína componente do complexo repressor Polycomb 2 (36% do input) sob condições suplementadas com andrógeno (+R1881). O lincRNA PVT1 também está associado fisicamente ao AR (10% de input) e à EZH2 (42% de input) em condições de privação de andrógeno (-R1881). Assim, a associação física entre lincRNA PVT1, AR e EZH2 é independente do hormônio andrógeno. Usando uma abordagem de estudo em larga-escala de perda e ganho de função, nossos resultados mostraram que o silenciamento do lincRNA PVT1 em células LNCaP na presença de andrógeno restaura a expressão parcialmente, totalmente ou causa superexpressão de 160 genes que tiveram a expressão inibida por andrógeno. Entre esses genes, destacamos genes envolvidos na regulação da diferenciação celular, em componentes da junção célula-célula, na inibição da migração e invasão celular e no desencadeamento da via apoptótica. Imunoprecipitação da cromatina seguida de PCR quantitativo (ChIP-qPCR), em cultura de células LNCaP suplementada com andrógeno sob silenciamento do lincRNA PVT1, mostrou aumento significativo na ocupação pela marca de histona ativadora H3K27Ac do promotor do gene NOV, um dos genes que tiveram sua expressão aumentada com o silenciamento de PVT1. O ChIP-qPCR também mostrou, após o silenciamento do lincRNA PVT1, um aumento significativo da marca H3K27me3 na região enhancer do gene NOV, uma característica de enhancers poised (prontos para ativação). Em conclusão, nós fornecemos a primeira evidência experimental para um mecanismo de ação do oncogene lincRNA PVT1 em células de câncer de próstata e demonstramos que sua ação inibidora da expressão afeta genes alvo que facilitam a proliferação e migração de células do câncer de próstata, sugerindo que o lincRNA PVT1 é um novo agente no complexo mecanismo de repressão transcricional envolvendo um RNA silenciador, o receptor de andrógeno (AR) e o potenciador de Zeste homólogo 2 (EZH2) no remodelamento da cromatina em células LNCaP

Long non-coding RNA (lncRNA) plasmacytoma variant translocation 1 (PVT1) is an oncogene known to be overexpressed in various types of cancer. PVT1 lincRNA is located in the wellknown cancer-related genomic region 8q24, also known as 'gene desert. PVT1 lincRNA level of expression is associated with increased prostate cancer (PCa) risk and is correlated with androgen receptor (AR) expression levels. However, the mechanism of PVT1 and AR involvement in the development of prostate cancer is still unclear. Here, we tested the hypothesis that formation of the complex AR-EZH2-PVT1 participates in the regulation of gene expression in prostate cancer, in LNCaP cells. Ribonucleoprotein immunoprecipitation followed by quantitative PCR (RIP-qPCR) revealed that PVT1 lincRNA binds both the AR (12 % of PVT1 input) and the methyltransferase EZH2 from the Polycomb repressive complex 2 (36 % of input) under androgen-supplemented conditions (+R1881). PVT1 also binds both AR (10 % of input) and EZH2 (42 % of input) under androgen-deprived conditions (-R1881). Thus, PVT1 binding to AR and EZH2 is independent of the androgen hormone. Using a large-scale loss and gain of function approach, our results show that PVT1 knockdown (KD) in LNCaP in the presence of androgen restores the expression partially, fully or causes overexpression of 160 genes that are inhibited by androgen. Among these genes, we highlight genes involved in regulation of cell differentiation, in components of cell-cell junction, in inhibition of cell migration and invasion and in triggering of the apoptotic pathway. Chromatin immunoprecipitation followed by quantitative PCR (ChIP-qPCR) with LNCaP cells in androgen-supplemented cultures under PVT1 lincRNA knockdown showed a significant increase in occupancy by the histone activation mark H3K27Ac of the promoter region of the NOV gene, one of the genes that had an increased expression upon PVT1 silencing. ChIPqPCR also showed a significant increase upon PVT1 lincRNA silencing of the H3K27me3 histone mark in the enhancer region of the NOV gene, a distinct feature of poised enhancers. In conclusion, we provide first experimental evidence for a mechanism of action of PVT1 lincRNA oncogene in prostate cancer cells, and show that its inhibitory action affects targetgenes that facilitate proliferation and migration of prostate cancer cells, thus suggesting PVT1 lincRNA as a novel lncRNA player in the complex mechanism of transcriptional repression involving a silencer RNA, the androgen receptor (AR) and the Enhancer of zeste homolog 2 (EZH2) in chromatin remodeling in LNCaP cells

Expression of plasma-cytoma variant translocation gene 1 in ovarian cancer and its malignant biologic behavior / 中国肿瘤生物治疗杂志

@#[Abstract] Objective: To investigate the effects of plasma-cytoma variant translocation gene 1 (PVT1) on proliferation, migration and invasion of ovarian cancer SKOV3 cells. Methods: Quantitative reverse transcription PCR (qRT-PCR) was used to detect the expression of PVT1 in 32 pairs of ovarian cancer tissues and corresponding para-carcinoma tissues. The effects of PVT1 on proliferation, migration and invasion of ovarian cancer cells were studied by CCK-8, scratch wound healing assay and Transwell assay. Results: The expression level of PVT1 in SKOV3 cells and ovarian cancer tissues was significantly increased (all P<0.01). The expression level of PVT1 was correlated with histological grade, FIGO stage and lymph node metastasis in patients with ovarian cancer (P<0.05 or P< 0.01).After transfection with PVT1 siRNAfor 36, 48 h, expression of PVT1 was significantly decreased in SKOV3 cells; and the inhibition of PVT1 expression could decrease the proliferation ability and inhibit the migration and invasion of SKOV3 cells (P<0.05 or 0.01). Conclusion: LncRNA PVT1 was highly expressed in ovarian cancer. Down-regulation of PVT1 could inhibit the proliferation, migration and invasion of ovarian cancer SKOV3 cells.

Effects of lncRNA PVT1 silencing on C666-1 cell proliferation,migration and invasion / 实用医学杂志

Objective To explore the effects of down-regulated PVT1 on the proliferation,migration and invasion of nasopharyngeal cancer(NPC)C666-1 cells. Methods Expression of PVT1 in NPC cell lines was de-tected by real-time RT-PCR. The effects of PVT1 silencing on cell proliferation were detected using MTT assay. Transwell assay was performed to assess the effect of down-regulated PVT1 on C666-1 cells migration and invasion. Protein level of N-cadherin,Vimentin and E-cadherin were determined by qRT-PCR and Western blot. Results Expression of PVT1 was significantly increased in seven NPC cell lines. Expression of PVT1 was reduced compared to the control group after the siPVT1 transfection. C666-1 cell proliferation was inhibited as compared with siNC group after transfection. The number of migration and invasion cells were significantly reduced while down-regulat-ed PVT1 expression. E-cadherin expression was upregulated while N-cadherin and Vimentinafter was downregulat-ed after PVT1 silencing. Conclusions Downregulation of PVT1 in C666-1 cells via siPVT1 transfection can inhib-it cell growth,migration and invasion,as well as reverse epithelial-mesenchymal transition. PVT1 may serve as a biomarker or even a therapeutic target for NPC.

Expression of hsa_circ_PVT1 in human hepatocellular carcinoma and its clinical significance / 解放军医学杂志

Objective To determine the expression and clinical significance of circ-PVT1 in human hepatocellular carcinoma (HCC) and its effect on HCC cell proliferation.Methods The expressions of circ-PVT1 in hepatocellular carcinoma and the matched tumor-adjacent tissues were detected by RT-qPCR and the relationship between pathological indexes and the expression level was analyzed in 46 patients.The expressions of circ-PVT1 in human normal liver cell line (L02) and hepatocellular carcinoma cell lines (HepG2,SMMC-7721,MHCC-97H,MHCC-97L,HCC-LM3) were detected by RT-qPCR and were compared thereafter.With knocking down the expression of circ-PVT1,si-circPVT1 was transfected into HepG2 and SMMC-7721 cells by using lipofectamine technique in vitro,with the si-NC being taken as negative control.After interfering the expression of circ-PVT1,the effect on the proliferation of hepatocellular carcinoma cells was detected by CCK-8 and EDU experiments and flow cytometry was conducted to observe the effect of circ-PVT1 on cell cycle.Results The expression level of circ-PVT1 was significantly higher in HCC tissues than in adjacent tissues (P＜0.01),and its high expression level was significantly correlated with tumor size,TNM stage and differentiation degree.Similarly,in human hepatocellular carcinoma cell lines (HepG2,SMMC-7721,MHCC-97H,MHCC-97L,HCC-LM3),the expression level of circ-PVT1 was also higher than that in human normal liver cell line L02 (P＜0.05).Compared with the negative control group,silencing of circ-PVT1 resulted in remarkable reduction in cell proliferation of HepG2 and SMMC-7721.Conclusion circ-PVT1 may act as a potential biomarker for HCC diagnosis and may become a novel proliferation factor.

Long non-coding RNA PVT1 regulates effect of miR-551 on migration and invasion abilities of ovarian cancer through Wnt signaling pathway / 中国病理生理杂志

AIM:To investigate the expression of long non-coding RNA PVT1 in ovarian cancer and the role of PVT1 in migration and invasion abilities of ovarian cancer cells.METHODS: The expression of PVT1 in ovarian cancer tissue,normal ovarian tissue and different ovarian cancer cell lines was detected by qPCR.Transwell assay was used to de-tect the invasion ability of ovarian cancer cells after PVT1 silencing.The migration ability of the ovarian cancer cells after PVT1 silencing was detected by scratch test.The interaction between PVT1 and microRNA(miR)-551 was analyzed by dual-luciferase reporter assay.The effect of miR-551-inhibitor on the invasion and migration abilities of ovarian cancer cells after PVT1 silencing was detected by Transwell assay and scratch test.The expression of Wnt signaling pathway-related pro-teins was determined by Western blot after PVT1 silencing.The effects of PVT1 silencing on tumor weight and volume of ovarian cancer were examined by subcutaneous tumor transplantation in nude mice.RESULTS:The expression of PVT1 in ovarian cancer tissue was significantly higher than that in normal ovarian tissue(P<0.05).The expression level of PVT1 in ovarian cancer cell line ES-2 was the highest.PVT1 silencing inhibited the invasion and migration abilities of the ovarian cancer cells.After PVT1 silencing,miR-551-inhibitor promoted the invasion and migration abilities of the ovarian cancer cells.The expression of Wnt signaling pathway-related proteins was decreased after PVT1 silencing(P<0.05).Compared with negative control group,the tumor volume and weight in PVT1-siRNA group were significantly decreased(P<0.05). CONCLUSION:PVT1 plays an important role in the development of ovarian cancer.PVT1 regulates the invasion and mi-gration abilities of ovarian cancer cells through Wnt signaling pathway.

Effect of LncRNA-PVT1 on Proliferation and Apoptosis of Human Pancreatic Cancer Cell Line HPAF-Ⅱ / 胃肠病学

Background:Recent studies have shown that long non-coding RNAs(lncRNAs)play important roles in carcinogenesis and cancer biology and the related context has attracted more and more attentions. PVT1,which encodes a lncRNA,is reported to be up-regulated and exhibit pro-oncogenic activity in a wide variety of human cancers. Aims:To investigate the expression of PVT1 in human pancreatic cancer cells and its effect on proliferation and apoptosis of HPAF-Ⅱ cells. Methods:One target siRNA against PVT1 was synthesized and transfected into HPAF-Ⅱ cells by using lipofactamine technique. PVT1 mRNA expression was detected by real-time PCR;capability of cell proliferation was examined by MTS and colony formation assays;cell cycle progression and apoptosis were measured by flow cytometry;and Western blotting was performed to determine the expressions of apoptosis-related proteins and proto-oncogene protein c-Myc. Results:The mRNA expression of PVT1 in several human pancreatic cancer cell lines,especially HPAF-Ⅱ cells was significantly higher than that in H6c7,a human immortalization normal pancreatic ductal epithelial cell line. Compared with HPAF-Ⅱ cells transfected with negative control siRNA or without transfection,silencing of PVT1 by siRNA-PVT1 resulted in remarkable reduction in cell proliferation,cell cycle G1 phase arrest,and notable apoptosis;meanwhile,the expressions of apoptosis-related proteins(cleaved-caspase-3 and cleaved-PARP)were up-regulated,the ratio for Bcl-2 / Bax was decreased,and the expression of c-Myc protein was down-regulated. Conclusions:LncRNA-PVT1 is highly expressed in human pancreatic cancer cell line HPAF-Ⅱ. It may affect the proliferation and apoptosis of HPAF-Ⅱ cells partially through regulating c-Myc expression.