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Lncrna-RMRP Promotes Proliferation, Migration and Invasion of Bladder Cancer Via Mir-206

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Lncrna-RMRP Promotes Proliferation, Migration and Invasion of Bladder Cancer Via Mir-206 European Review for Medical and Pharmacological Sciences 2019; 23: 1012-1021 lncRNA-RMRP promotes proliferation, migration and invasion of bladder cancer via miR-206 H.-L. CAO1, Z.-J. LIU2, P.-L. HUANG1, Y.-L. YUE3, J.-N. XI1 1Beijing Rehabilitation Hospital Affiliated to Capital Medical University, Beijing, China 2Shandong Provincial Western Hospital, Jinan, China 3Liaocheng People’s Hospital, Liaocheng, China Abstract. – OBJECTIVE: The incidence of an and American countries1. Recently, it has in- bladder cancer (BC) is common in the world, but creased its morbidity and rejuvenation. BC risk its detail mechanisms for occurrence and devel- factors are smoking and long-term exposure to opment remain unclear. Recently, long non-cod- industrial chemical products, which are related ing RNAs (lncRNAs) have been observed to play an important role in many different diseases. In to the changes in the gene level of patients; more- this research, we mainly explored the role of the over, the variety of apparent heredity is involved RNA component of mitochondrial RNA process- in the development of tumors2. The occurrence ing endoribonuclease (lncRNA-RMRP) in blad- and development of bladder cancer is a chronic der cancer. process with multi-factors and multi-steps3. Thus, MATERIALS AND METHODS: We used qRT- investigating the detail mechanisms involved in PCR to detect the expression of lncRNA-RMRP in bladder cancer patients and tumor cells, and BC bladder cancer will provide a novel strategy the clinical significance was also analyzed. The for its prevention and treatment. Long nonco- methyl thiazolyl tetrazolium (MTT) assay was ding RNAs (lncRNAs) are new members in the used to detect the cell proliferation, and we used oncology field, containing 200 nucleotide (nt)- transwell to detect the migration and invasion, long RNA molecules and stably existing in the after the lncRNA RMRP was inhibited. West- plasma and urine, with disease and tissue speci- ern-blot was used to measure the relative pro- ficity and with no protein-coding potential4-6. At tein expression level in bladder cancer cells af- ter transfection with siRNA-NC or siRNA-RMRP. the present, they are being studied to suggest a RESULTS: We found that the lncRNA RMRP potentially pivotal role in tumor occurrence and was highly expressed in bladder cancer tissue, inhibition pathways7,8. The lncRNAs are able to compared with adjacent tissue. We also found identify complementary sequences, and this par- that the expression of RMRP was closely related ticular role is important to RNA in the post-tran- with the size, lymph node metastasis and surviv- scription, which involves cutting, editing, tran- al time of patients. What’s more, RMRP could pro- 9,10 11 mote the proliferation, migration and invasion of sport and translation . Wang et al found that BC cell lines via regulating miR-206 as a sponge. lncRNA-HULC can be used as molecular bait, CONCLUSIONS: According to the results, we or can be called miRNA sponge, which can in- found that lncRNA RMRP was closely related to fluence the expression of target protein RPKACB the progression of bladder cancer, which could by regulating the expression of miR-372 in blad- be a potential target for treating BC patients. der cancer. Moreover, Yuan et al12 suggested that Key Words: TGF-beta promoted the expression of lncRNA-A- Bladder cancer, miR-206, lncRNA-RMRP, Migration, TB in hepatocytes, which could play the function Invasion, Proliferation. of microRNA sponge. It could be specific to the combination of miR-200 family, thus inhibiting miR-200s as the function of anticancer microR- NA, improving significantly the expression of Introduction CDK9/2, which is target gene of miR-200s, and promoting the migration of bladder cancer12. The Bladder cancer (BC) is the most common RNA component of mitochondrial RNA pro- malignant tumor in the urinary system and it is cessing endoribonuclease (RMRP), a lncRNA, the fourth of male malignant tumors in Europe- was first discovered in cartilage-hair hypoplasia 1012 Corresponding Author: Jianing Xi, MD; e-mail: [email protected] lncRNA-RMRP promotes proliferation, migration and invasion of bladder cancer via miR-206 (CHH), an autosomal recessive inherited disea- titative PCR System (Applied Biosystems, Foster se13. Recently, some studies also found that lncR- City, CA, USA) according to the manufacturer’s NA-RMRP played a very important role in tumor. instructions. The GAPDH was used as a referen- Shao et al14 demonstrated that lncRNA-RMRP ce gene. The PCR primers for RMRP, glyceral- play a crucial role in the occurrence and pro- dehyde-3-phosphate dehydrogenase (GAPDH) gression of gastric cancer by acting as a miR-206 were as follows: RMRP, 5ʹ-ACTCCAAAGTCC- sponge. However, whether the lncRNA-RMRP GCCAAGA-3ʹ and 5ʹ-TGC GTAACTAGAGG- executed its function in bladder cancer remains GAGCTGAC-3ʹ; GAPDH, 5ʹ-ACCCACTCC- largely unclear. In this work, we aimed to detect TCCACCTTTGAC-3ʹ and 5ʹ-TGTTGCTGTAG the expression of lncRNA RMRP in the bladder CCAAATTCGTT-3ʹ. All experiments were repe- cancer and explored the possible mechanism of ated at least three times. lncRNA RMRP affecting the malignant activity of bladder cancer. CCK8 Assays The Cell Counting Kit-8 (Beyotime Inst. Bio- tech, Shanghai, China) was used to determine the Patients and Methods cell proliferation, according to the manufactu- rer’s instructions. Three replicate wells were set Patients in each group. CCK8 assay was done as follows: 91 cases of bladder cancer (BC) tissue and the 96-well flat-bottomed plate was used to seed adjacent tissue were collected from Beijing Reha- 6×10 3 cells/well, and grown at 37°C for 24 h, then bilitation Hospital Affiliated to Capital Medical transfected with corresponding vector. Finally, University in Beijing City (Beijing, China) from the absorbance was determined at 450 nm using a May 2016 to June 2017. All of the patients were microplate reader (Bio-Rad, Hercules, CA, USA). well informed and informed consents were also The data were collected for 3 days. All experi- signed. The tissues were divided into equal size ments were repeated at least three times. and frozen in liquid nitrogen after surgery. The experiment protocol was approved by the Institu- Transwell Assay tional Review Board of the Beijing Rehabilitation The transwell insert (8 μm, Corning, Corning, Hospital Affiliated to Capital Medical University NY, USA) was used to determine the invasion BC (Beijing, China). cells. After 24 h transfection, 5 × 104 cells were first starved in 200 ml serum free medium and Cell Culture then seeded in the dishes. The lower chamber Normal urothelial cell line SV-HUC-1 and was filled with 500 ml of completed medium. We bladder cancer cells BIU-87, T24 were purcha- incubated the cells for 48 h at 37°C, which had sed from the Institute of Cell Research, Chinese migrated to the bottom surface of the filter mem- Academy of Sciences (Shanghai, China). The SV- brane; next, they were stained with 0.5% crystal HUC-1 and T24 cells were cultured in Dulbecco’s violet solution and photographed. At last, the ab- Modified Eagle Medium (DMEM) plus 10% fetal sorbance was determined at a wavelength of 570 bovine serum (FBS, Invitrogen, Carlsbad, CA, nm using a microplate reader (Bio-Rad, Hercules, USA). The BIU-87 was cultured in Roswell Park CA, USA). The whole experiments were repeated Memorial Institute-1640 (RPMI-1640) Medium at least three times. plus 10% fetal bovine serum (FBS, Invitrogen, Carlsbad, CA, USA). All cell lines were placed at Luciferase Assays 37°C with a humidified atmosphere of 5% CO2 in Wt-RMRP/mut-RMRP sequences were ampli- incubator. fied and cloned into the downstream of the stop codon of the firefly luciferase in basic vector (Pro- RNA Extraction and Real-Time mega, Madison, WI, USA). Quantitative PCR Assays Total RNA was extracted from HEK293 cells, Total RNA from tissue samples and transfected which was reverse transcribed into cDNA. The cells was extracted by using TRIzol reagent (In- potential binding sites of pmiR-RMRP-WT and vitrogen, Carlsbad, CA, USA), following the mutant sequence pmiR-RMRP-MT were synthe- manufacturer’s protocol. Reverse transcription sized into pmiR-GLO (Promega, Madison, WI, polymerase chain reaction (RT-PCR) was perfor- USA). After that, miR-206 mimics and miR-206 med using ABI PRISM 7000 Fluorescent Quan- negative control (NC) were co-transfected into 1013 H.-L. Cao, Z.-J. Liu, P.-L. Huang, Y.-L. Yue, J.-N. Xi HEK293 cells with pmiR-GLO for 24 h. Renilla neChip Human Gene 2.0 ST Array to scanning expression vector was transfected into each group fluorescence. to serve as a normalized control. 48 h after tran- Using small sample statistical learning and se- sfection, firefly and Renilla luciferase activities condary test of random sampling, we found that were measured using Dual Luciferase Reporter RMRP was highly expressed in heatmap of blad- Assay System (Promega, Madison, WI, USA). der cancer (Figure 1A). Furthermore, we used qRT-PCR to detect the expression of RMRP in Western Blot Assays BC tissues and adjacent tissues (Figure 1B). The Protein was extracted by RIPA lysis buf- results showed that RMRP was high expressed in fer containing phenylmethylsulfonyl fluoride BC tissues compared with adjacent tissues. (PMSF) (Beyotime Biotechnology, Shanghai, China). The concentration of protein was detected The Clinical Characteristic of RMRP by the standard bovine serum albumin (BSA) In order to explore the clinical significance protein quantitation assay. 60 mg of samples were expression of RMRP in BC, we investigated the added to sodium dodecyl sulfate polyacrylamide clinical characteristic of RMRP. We measured gel electrophoresis (SDS-PAGE) on a 10% dena- the expression level of RMRP from the clinic pa- turing gel. The protein was transferred to poly- thological information of the patients.
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