Functions And Mechanistic Studies Of LSINCT5 In The Regulation Of Malignant Progress In Epithelial Ovarian Cancer

Epithelial ovarian cancer(EOC)is one of the most frequent malignant tumors of female reproductive system.The incidence of the disease is the third in the female reproductive system malignancies,but the mortality rate is arranged first.With the accelerated aging of China's society,its incidence in the world has recently been increasing.Lack of specific symptoms and effective diagnostic,about 70% of the patients with ovarian cancer have occurred abdominal cavity or distant metastasis for the first time diagnosis,the five year survival rate is only about 30%.The high mortality rate is mainly related to ovarian cancer metastasis.Therefore,the study on the mechanism of metastasis and invasion of ovarian cancer is the hotspot flush and difficult.Long chain non coded RNA(Inc RNA)is a newly discovered class of non coded RNA in recent years.More than 200 nucleotides in length.In the past,Inc RNA was often considered "noise" and had no biological function,but some studies have shown that lnc RNA involved in the epigenetics,transcription activation,post transcriptional regulation and other important regulation,playing a significant role in many biological processes,including genomicimprinting/protein/chromosomesilencing/transcriptionactivation/chromatin modification/transcrip-tion/interference/activity regulation of nuclear transport,and participate in a variety of human diseases,especially tumor progression,metastasis and deteriorationRecent studies have shown that lnc RNAs is also an important regulatory factor for cell stress to regulate cell response.Stress induced lnc RNAs is closely related to cancer invasion and metastasis.Recent studies have proved that LSINCT5 is a long stress-induced non-coding transcript.It is a normal human bronchial epithelial cells(NHBE)receiving amino-1-methyl nitrite in tobacco(3-pyridyl)(NNK)stimulation.LSINCT5 is highly expressed in breast and gastric cancer,and is significantly related to the prognosis of gastric cancer.Silva and other studies have confirmed that the expression of LSINCT5 in breast and ovarian cancer significantly increases,and can promote the proliferation of malignant tumor cells.However,the relationship between the expression of LSINCT5 and the clinicopathological relationship and its role in the invasion /metastasis of ovarian cancer remains unclear.In this study,we will study the expression of LSINCT5 in epithelial ovarian cancer cells and tissues,and analyze its relationship with clinicopathology and prognosis.To explore the role and mechanism of invasion / metastasis.Part one Expression and prognosis of LSINCT5 in ovarian cancer1 ObjectiveTo study the expression of LSINCT5 in the tissues and cells of epithelial ovarian cancer,and to analyze the relationship between the clinicopathology and the prognosis.2 Methods2.1 RNAs were extracted from 40 tissue samples of EOC and 30 samples of normal ovarian surface epithelial tissue using TRIzol.The LSINCT5 expression level between the two were detected by quantitativereal-time PCR(q PCR);LSINCT5 expression was examined in SKOV3,OVCAR3 and 3AO cells by q PCR.2.2 The relationship between LSINCT5 expression and clinical pathology(age,FIGO stage,pathological grade,histological type,size of residual lesion,lymph node metastasis)were analyzed by q PCR;The specificity and sensitivity of ovarian cancer were diagnosed by LSINCT5 curve(ROC curve)2.3 To investigate the relationship between the expression levels of LSINCT5 and the prognosis of patients with EOC by survival analysis.3 Result3.1 The q PCR showed the LSINCT5 expression was largely elevated in EOC tissues compared to control normal tissues(P<0.01).3.2 The expression of LSICT5 in the most aggressive SKOV3 cells were significantly up-regulated compared with OVCAR3 and 3AO cells(P<0.05).3.3 High LSINCT5 expression levels were positively correlated withthe FIGO stage,the occurrenceof lymph node metastasis(P<0.05),but not with histological grade of the tumor,the age of the patient,the histological type of the tumor and the residual tumor diameter;The area was 0.927 under the ROC curve.The optimum critical point was 23.7133,the sensitivity was 80%,and the specificity was 93%.3.4 In a univariate analysis,LSINCT5 expression levels were correlated with PFS(P=0.028).According to the multivariate Cox-regression analysis,LSINCT5 expression levels,the FIGO stage were independent predictors of PFS(95%CI 0.53-1.116).FIGO stage(95%CI 0.570-13.426)and residual lesionsize(95%CI 0.220-2.77)were independent prognostic factors for OS in patients with ovarian cancer.The risk of death in stage III patients was 4.295 times higher than those in stage I and II patients.The risk of death in dissatisfied patients was satisfactory 1.593 times.4 ConclusionLSINCT5 may be involved in the invasion and metastasis of ovarian cancer and influence the prognosis.Part two The study of LSINCT5 promote the malignant biological behavior of ovarian carcinoma1 ObjectiveTo study the malignant biological behavior of epithelial ovarian cancer after silencing LSINCT5.2 Methods2.1LSINCT5 was knocked down by gene silencing,and si-LSINCT5 was transfected into SKOV3 cells,which has the highest transfection efficiency was screened.2.2 The effect of si-LSINCT5 on the proliferation of SKOV3 cells was assessed using the Cell Counting Kit-8(CCK-8);Cellapoptosis were assessed using flow cytometry;The cellinvasion and metastasis were assessed usingcell scratch test and transwell invasion test.3 Results3.1Theinterference efficiency of LSINCT5-si RNA-1 sequence was significantlyhigher than LSINCT5-si RNA-2(P<0.001),and LSINCT5-si RNA-1 was selected as the follow-up interference sequence.3.2 CCK-8 assay revealed that si-LSINCT5 significantly reduced the growth rates of SKOV3 cells compared with NC groups(P<0.05);Flow cytometry assay revealed that there was no obvious change in apoptosis(P>0.05).The wound-healing assays showed si-LSINCT5 in SKOV3 cells restrained the cell migration compared with NC(P<0.05).Similarly,si-LSINCT5 significantly decreased invasion of SKOV3 cells compared with NC(P<0.05).4 ConclusionSilence LSINCT5 can inhibit ovarian cancer SKOV3 cells malignant biological behavior.Part three The mechanism of LSINCT5 regulating the malignant progression of ovarian cancer cells1 ObjectiveTo explore the molecular mechanisms of LSINCT5 regulated ovarian malignant biological behavior.2 Methods2.1 To explore thegene expression for si-LSINCT5 SKOV3 cells by chip technology,and the target gene with bioinformatics analysis.2.2 Detection of CXCR4 gene and protein expression for si-LSINCT5 SKOV3 cells with PCR and Western blot.2.3 QPCR was used to detect the correlation between LSINCT5 expression andCXCR4 in EOC.2.4CCK8 cell proliferation test,cell scratch test and Transwell invasion test were used to detect the changes of proliferation,invasion and metastasis after adding CXCL12 factor.3 Results3.1 Gene expression profiles in si-LSINCT5 and NC SKOV3 cells was performed.Of the 784 genes examined by Whole Genome DNA array(Flod change?2,P<0.05),differentially expressed were 159 and 625 were up-regulated and down-regulated,respectively.The target gene of LSINCT5 was predicted,the effect of Cis was 0,and the effect of trans was 248.Meeting both more than2 times chanage and the target gene were CXCR4.3.2 Compared with the NC group,after silencing LSINCT5,the CXCR4 gene and protein expression decreased significantly(P<0.05)3.3 In ovarian cancer tissues,the positive rate of CXCR4 expression was 80%(32/40),which was significantly higher than that of normal ovarian tissue 13.3%(4/30)(P<0.01).Immunohistochemical staining was mainly located in the cytoplasm.The relationship between CXCR4 expression and age,FIGO staging,tissue differentiation and lymph node metastasis was analyzed.The results showed that the expression of CXCR4 in lymph node positive patients was significantly higher than that of negative(P<0.01);Thepatients with high-expression of LSINCT5 was significantly higher than that of low expression(P<0.05),while the age,tissue differentiation and FIGO were not significantly.(P>0.05).3.4 In ovarian cancer,the expression of LSINCT5 was positively correlated with CXCR4(r=0.613,P=0.00).3.5In si-LSINCT5 SKOV3 cell culture medium,CXCL12 was added,and cellproliferation ability increased(P<0.05).The invasion and metastasis ability increased(P<0.05).Conclusion: LSINCT5 regulates the proliferation,invasion and metastasis of ovarian cancer cells by regulating the CXCR4/CXCL12 signal axis,and is a potential target for treatment of ovarian cancer cells.