| Background and ObjectiveThe incidence of cervical cancer ranks second in the numberof female cancer deaths,second only to breast cancer,with high morbidity and mortality.Finding new therapeutic targets and prognostic markers is an urgent need to improve the survival rate of cervical cancer patients.Many non coding RNAs(lncRNAs)are key regulators in various biological processes.Their abnormal expression is closely related to the progression of cervical cancer.Long non coding RNAs(lncRNAs)is a kind of intracellular RNA,which does not encode protein,but plays an important role in regulating cell activities such as chromosome silencing,genomic imprinting,chromatin modification,transcription activation,post transcriptional regulation and protein regulation.According to published papers,lncRNAs plays an important role in the occurrence and development of various kinds of human tumors.LncRNAs plays an important role in the development of various kinds of human tumors.HOXD is a member of the homeobox(Hox)gene family and plays a crucial role in the development of embryos and organs.HOXD cluster antisense RNA 1(HOXD AS I)is a new LncRNA,which is transcribed from the HOXD gene cluster of human chromosome 2q31.2 in an antisense manner.More and more evidence suggests that ectopic expression of HOXD-AS 1 is involved in the development and progression of various types of human tumors.Such as Hepatocellular carcinoma,bladder cancer,prostate cancer,neuroblastoma and gastric cancer,etc.The relationship between hoxd-as1 and cervical cancer is unclear.We’Ve consulted NONCODE Scientific database(http://www.Noncode.org/),A comprehensive data platform for researchers to analyze non-coding RNA genes was developed and maintained by bioinformatics research group of cas institute of computing technology and bioinformatics laboratory of cas institute of biophysics.we We found that hoxd-asl is an abnormal expression of LncRNAs related to cervical tissue.We took hoxd-asl as the research object,and determined the relationship between hoxd-asl and cervical cancer by detecting the expression level of hoxd-asl in uterine tissues and cells,and explored the specific role of hoxd-asl in cervical cancer and its mechanism through subsequent in vitro experiments.Methods1.Patients and specimensA total of 122 cervical cancer surgical specimens collected from XXXX hospital between 2013 and 2015 were placed in the frozen storage tube after excision,and the sample information was marked and stored in liquid nitrogen.The whole process of preservation and transport was conducted in accordance with the principle of no enzyme.These cases were not preoperatively treated with chemotherapy,radiotherapy and chemotherapy.Detailed pathological features and clinical data were obtained by retrospective analysis of complete archived medical history,including age,tissue type,degree of differentiation,clinical stage,whether lymphatic vascular interstitial infiltration,lymph node metastasis,and complete medical records.Follow-up was used to determine whether patients could relapse.The acquisition and operation of specimens conform to the ethical standards and operating procedures of clinical trials.2.Cell lines and culture methodsFive types of cervical cancer cell lines(HeLa,CaSki,me-100,C33A and SiHa)and normal human cervical cell lines HOSE were obtained from American Type Culture Collection(ATCC,Manassas,VA,USA),and were cultivated and passed down by our laboratory.The cells were cultured in rpm-1640 medium containing 10%fetal bovine serum and 100UI/mL penicillin and 100ug/mL streptococcus enzyme.50 ml of cell culture bottle at 37 ℃ in 5%C02 in cultivation in the Incubator3.Real-time fluorescence quantitative PCRRNA was extracted from tissue and cell samples by Trizol.Rt-pcr kit(Takara)was used to transcribe RNA into cDNA according to the instructions.Sequences of hoxdas1 and U6 primers were obtained from TaKaRa.U6,as the internal reference,was prepared into the mixed liquid by 20ul system and put into abi7,500 instrument to test the expression of hoxd-asl in cervical tissues,five kinds of cervical cells and normal cervical cell lines.The data mean of three independent experiments was taken as the result.4.Cells transfectionsiRNA(hoxd-as siRNAl and hoxd-asl siRNA2)for hoxd-asl and hoxd-as1 minics and negative control group(control group)were obtained from gemma pharmac Cells in the logarithmic growth stage were inoculated in a 6-well plate and observed 14 days after inoculation.The experiment was terminated when cell cloning eutical co.,LTD(Shanghai,China).After adhering to the 6-well plate,HeLa and CaSki cells were transfected with Lipofectamine 2000.According to the preset group,rt-pcr was used to detect the expression of hoxd-as1,and subsequent experiments were conducted.5.Cell proliferation assayAfter transfection,6hrs,12hrs,24hrs,48hrs and 72hrs were inoculated on 96-well plates,and Cell Counting kit-8 Kit was used to detect the Cell viability.The absorption value of each group of samples was measured at the wavelength of 450nm and the growth curve was drawn.6.Coloniy formation assayCells in the logarithmic growth stage were inoculated in a 6-well plate and observed 14 days after inoculation.The experiment was terminated when cell cloning was visible to the naked eye.0.1%crystal violet staining was used to calculate the clone formation rate.7.Cell scratch testThe cells were transfected 24 hours later,and when the cells grew to 100 percent confluence,they were plotted perpendicular to the plate.The exfoliated cells were rinsed and cultured in 37 medium for 24 hours,examined under microscope every 6h,and photographed and recorded,and the cell growth area coverage ratio was calculated.8.In vitro invasion experimentAfter 24 hours of transfection,membrane enzyme was used to digest and make a single cell suspension,and the density of 1 x 105 cells per hole was inoculated in the upper chamber of the 24 hole plate used by Transwell,and 200 ul of suspension was added to each hole in the 24 hole plate.500 ul medium was added to the next chamber of the hole,and after 24h of culture,0.1%crystal violet was stained.The cell attachment to the basement membrane of Transwell chamber was observed and photographed by forward microscope.Five random fields were selected for counting and cell invasion rate was calculated.9.Westen blot analysisAfter cell/tissue protein extraction,the protein concentration was measured by BCA.SDS-PAGS electrophoresis was performed with 10%separation gel.The film was scanned and the optical density and molecular weight of the strip were analyzed with the gel image processing system to complete the statistical analysis.10.Tumor formation in nude miceHeLa cells transfected with siRNA were treated as the treatment group,and untreated HeLa cells were treated as the control group.Suspension with cell concentration of 5 x 107/ml was prepared and injected into the left armpit of BALB/C nude mice at 4 weeks old,subcutaneously,each nude mouse was inoculated with 0.2ml.The length and short diameter of the tumor were measured by vernier caliper on the second day after tumor formation.The nude mice were sacrificed after 28 days,the tumor was dissected and weighed electronically.The weight of the tumor formation and tumor volume were compared in the two groups of experimen.11.immunohistochemical analysisThe tumor tissue was placed into a paraffin embedding tank for paraffin embedding and excised.Disinfect the slides with 75%alcohol and wipe them clean.Place the slides vertically in the incubator,so that the tissue slices are closely attached to the slides.As far as possible avoid tissue wrinkle and interlayer bubble formation.Dewaxing tissue sections 10 Minutes.The slides were divided into negative control group and positive control group according to the preset group.One antibody was applied on the slide successively.Second antibody was added with a drop of streptomycin anti-biotin-peroxidase solution on the slide,and the pre-prepared DAB display solution was added after completion.The solution was placed horizontally at room temperature for 6 minutes and observed under inverted microscope.DAB display solution was removed and stained with hematoxylin for 10-15 seconds.After completion,hydrochloric acid alcohol differentiation was immediately completed and ammonia water returned blue.The slides were dehydrated and dried with ethanol according to the preset etl gradient and action time.Look under the microscope.12.Statistical analysisSPSS20.0 software was used for statistical analysis of the measured data intake jin.All the test data were repeated at least 3 times,represented by X±S The comparison between the two groups of counting data was performed with bilateral t test,and the comparison of multiple groups of data was performed with multifactor analysis of variance,and the difference was taken(P<0.05)as statistically significant.Results1.The results of Rt-pcr tissue test:by detecting the expression of hoxd-asl in 122 cervical cancer cell specimens,we found that the expression level of hoxd-as1 in cervical cancer patients was correlated with tumor lymph node metastasis,clinical staging,vascular invasion,lymph node metastasis and tumor recurrence(P<0.05).There was no significant correlation with age and tissue differentiation(P>0.05).2.The results of Rt-pcr cell detection test:the expression of hoxd-as1 in five types of cervical cancer cells(HeLa,CaSki,me-100,C33Aand SiHa)and normal cervical cell line HOSE was detected by real-time fluorescent quantitative PCR.It was found that the expression level of hoxd-as1 in cervical cancer cells was higher than that in normal cervical cells(P<0.01).Among them,the expression of hoxd-asl in the mesenter:ic metastatic cells(CaSki)of epithelial cervical cancer(Hela)and epidermoid cystic cervical cancer was relatively high.3.The effects on the cervical cancer cells proliferation,migration and invasion After down-regulating and up-regulating the expression of hoxd-asl in cervical cancer cells:After hoxd-asl siRNA was transfected to down-regulate the expression of hoxd-as1 in cervical cancer cells,MTT experiments,scratch experiments,Transwell cell migration and cell invasion experiments proved that down-regulation of hoxd-asl expression can reduce the proliferation rate,self-enhancement,migration and invasion of cervical cancer cells.The proliferation rate,self-enhancement,migration and invasion of cervical cancer cells were enhanced after the expression of hoxd-asl was up-regulated.4.The Results of West blotting,it was found that the p-erk and Ras protein levels of cervical cancer cells were significantly decreased after downregulation of hoxd-asl expression.After up-regulation of hoxd-asl,p-erk and Ras protein levels were significantly increased.5.Effects of erkl/2 inhibitors on up-regulated hoxd-as.l expression cells:after up-regulation of hoxd-as1 expression,we used erk1/2 inhibitors to inhibit the activation of Ras/erkl/2 pathway,and the results showed that the proliferation,migration and invasion of cervical cancer cells promoted by hoxd-asl minics were significantly inhibited after the effect of erkl/2 inhibitors.6.Tumor formation in nude mice:the tumor volume and weight of cervical cancer cells treated with hoxd-as1 siRNA were significantly smaller than those of the control group(P<0.01).7.Erkl/2 and Ras protein levels of tumor tissues in mice:western blot and immunohistochemistry showed that down-regulation of hoxd-asl expression significantly inhibited erkl/2 and Ras protein levels in tumor tissues in nude mice(P<0.01).Conclusion:1.The expression level of hoxd-asl in patients with cervical cancer was positively correlated with tumor clinical staging,vascular invasion,lymph node metastasis and tumor recurrence.2.Up-regulation of hoxd-asl expression of cervical cancer cells can promote their proliferation,self-renewal,migration and invasion abilities;Downregulation of hoxd-asl expression can inhibit proliferation,self-renewal,migration and invasion of cervical cancer cells.3.Hoxd-asl affects the ability to promote proliferation,self-renewal,migration and invasion of cervical cancer cells through the ERK/Ras pathway.4.Down-regulation of hoxd-asl expression of cervical cancer cells can inhibit tumor formation in nude mice,and its effect is achieved through the ERK/Ras signaling pathway,which is consistent with the results of in vitro cell experiments. |
PDF Full Text Request