Study On The Role And Mechanism Of MNX1-AS1 In The Development And Progression Of Ovarian Cancer

BackgroundOvary is an important reproductive organ of women,and plays an important role in maintaining secondary sex and reproduction.Ovarian Cancer(OC)is one of the malignant tumors in reproductive system which seriously endangers women’s life.Due to its early symptoms hidden,only 20%were found in the early stage of ovarian cancer.The mortality rate is the highest among gynecological tumors.Many patients have abdominal metastases before symptoms appear.the 5-year survival rate of advanced ovarian cancer patients is only 10-20%,and the 5-year survival rate of early diagnosis is as high as 85-90%.early symptoms hidden,poor prognosis and rising incidence rate in recent years has aroused wide concern in the academic circles.The most important factor in determining prognosis is whether there is metastasis,so it is significant to detect the lesion early and whether there is metastasis.1:long non-coding RNA(LncRNA)there are a lot of antisense,overlapping,non-coding RNAs(noncoding RNAs,NcRNA)in addition to about 20000 protein-coding genes in Human genome,initially,these non-coding transcription product is considered to be "noise"transcription,have no function,did not cause the attention of researchers.Recent years it is proved that these noncoding RNAs actually play an important role in the process of cell growth and metabolism.expecially long noncoding RNAs(IncRNAs)which exist in eukaryotes have attracted researchers’s attention.Their expression is closely related to the development and organism of diseases and tumors.2:The correlations between IncRNAs and ovarian cancer.There have been some researches on the correlations between IncRNAs with ovarian cancer,and made some achievements,H19,the IncRNA gene,related to gene imprinting,is product of IGF2(Insulin-like growth factor 2)gene,only expressed in embryonic tissue and not expressed in adult tissues.abnormally IGF2/H19 expressed in ovarian cancer,colon cancer,prostate cancer and other tumors.XIST genes are on the X chromosome inactivation centre,its XIST RNA transcription products play an important role in the female X chromosome inactivation,the intracellular XIST levelis lower in ovarian cancer epithelial cells than that in normal ovarian epithelial cells.and missing barr body may be due to the XIST Xi loss as a result of low level XIST to promote the occurrence of ovarian cancer.The sensitivity of ovarian cancer cells to the chemotherapy of paclitaxel inhibited XIST RNA expression,suggesting that it could be used as an evaluation marker for chemotherapy in ovarian cancer.In addition to the correlations between excessive HOTAIR gene expression with breast cancer,tumors HOTAIR expression level is negatively related to the level of ovarian cancer differentiation,with poor prognosis and low disease-free surial associated with lymph node metastasis.Suppressed HOTAIR can reduce the transfer ability of ovarian epithelial carcinoma,and suppresses the peritoneal metastasis of ovarian cancer in animals,speculated that they are affected by MMP-3 and MMP-9 and epithelial cell interstitial pathways.3:Lnc MNX1-AS1Lnc MNX1-AS1 is a non coding gene,located on chromosome 7,non-coding RNA transcription can generate 992 bp,MNX1-AS1 formal name is MNX1 antisense RNA 1(head to head),MNX1-AS1 on NCBI sequence address is https://www.ncbi.nlm.nih.gov/gene/645249.it has a another name-CCAT-5.CCAT(colon cancer associated transcript 1):colon cancer related transcription factors are the first class of long-chain non-coding RNAs found in colon cancer,according to the order of discovery,named CCAT-1,CCAT-2,CCAT-3,CCAT-4,and CCAT-5.In recent years,the relationship between LncRNA(CCAT)and tumor development has been reported,suggesting that LncRNA CCAT1 is related to the occurrence,development and prognosis of HCC,and is closely related to the development of gallbladder cancer..There are very little reports about Lnc MNX1-AS1,only reported an exceptionally high expression in gallbladder cancer.its expression network related to 23 IncRNA and 39 and mRNA,including such as trim59,otx1,HOXB9,Semac tumor associated protein coding genes,suggest its relations with gallbladder disease.Ye Z Zhou M and other reports believe that it is also closely related to the development of colorectal cancer.There is no report on the correlations between Lnc MNX1-AS1(CCAT-5)and ovarian cancer so far.In this study,the role and mechanism of Lnc MNX1-AS1(CCAT-5)in the role and mechanism of ovarian cancer are discussed.Part 1 The corresponding correlations between MNX1-AS1 expression and clinical pathology informaionObjective:1.The differences in expression of MNX1-AS1 in normal tissues and in ovarian cancer tissues2 analyse the correlations between MNX1-AS1 expression in ovarian cancer tissues with clinical pathologic factors(including age,pathologic differentiation degree,clinical stage and the correlation between lymph node metastasis).Methods:20 cases of pathologically diagnosed ovarian cancer sammples of qilu hospital of shandong university in June 2016 to June 2017 were collected.8 cases were under the 40 years old.(including 40),12 cases were over 40 years old.The high pathological differentiation group were 4 cases,5 cases of middle differentiation and 11 cases of low differentiation.Combined with FIGO(International Federation of Gynecology and Obstetrics,International Federation of Obstetrics and Gynecology),2015 clinical staging diagnostic criteria,15 cases of I and II patients,5 cases of stage III and IV patients;12 cases with lymph node metastasis and the remaining 8 patients with no lymph node metastasis.The control group was a normal tissue around 3cm outside the ovarian cancer tissue of the patient,and no invasion and metastasis.in the nomal tissures by histopathological diagnosis.Using real-time fluorescence quantitative PCR assay,the product was analyzed by Agrose electrophoresis,and the GraphPrism 5 statistical software was used,and the difference was found to be significant by 2-Ct scanning and p-value<0.05.Results:1.The clinical significance of MNX1-AS1 expression in ovarian cancerThe results of QRT-PCR showed that the expression level of MNX1-AS1 in ovarian cancer tissues was significantly higher than that in normal tissues(P<0.001).2.Correlations between the expression level of MNX1-AS1 and the clinical pathological factors of ovarian cancer patients.Low positive expression of MNX1-AS1 in Ovarian cancer tissues(40 years old and under the age of 40 years old)patients were 8 cases,high level positive expression was 0 case;low positive expression of MNX1-AS1 in Ovarian cancer tissues(above 40 years old)were 9 cases,high level of positive expression were 3 cases;after chi-square statistics analysis,there was no significant correlations(P>0.01)between MNX1-AS1 expression and patient years.According to the criteria of FIGO,the MNX1-AS1 high expression in patients with stage Ⅰ and Ⅱpatients were 2 cases,the other 13 cases were low expression.Among patients Ⅲand IV,was 1 cases of high expression with 4 cases of low expression.Chi-square test results showed that the expression of MNX1-AS1 in ovarian cancer tissue was significantly correlated with clinical staging(p<0.05).According to the degree of pathological differentiation,there were 10 patients with low expression of MNX1-AS1 in the samples of low differentiation of ovarian cancer,and 1 case of high expression.low expression of MNX1-AS1 was in 4 cases and high expression in 1 case in middle differentiation.1 case high expression,3 cases low expression in High differentiation group.The results showed that the pathologic differentiation was not significantly correlated with the expression level of MNX1-AS1 in ovarian cancer tissue.Among the 8 patients with no lymph node metastasis,8 of them were all low expression of MNX1-AS1.Among the 12 patients with lymph node metastasis,9 of them were MNX1-AS1 low expression and 3 high expression.Statistical analysis showed that MNX1-AS1 was significantly correlated with lymph node metastasis(p<0.05).Conclusions:1 The high expression of MNX1-AS1 in ovarian cancer was not significantly correlated with age and pathological differentiation(P>0.05).2 2.The high expression of MNX1-AS1 in ovarian cancer was significantlycorrelated with clinical staging of ovarian cancer and lymph node metastasis(P<0.05).3.MNX1-AS1 can be a biomolecular indicator for clinical staging of ovariancancer and to determine whether there is lymphatic metastasis.Part 2 The corresponding relationship between MNX1-AS1 expression and proliferation,apoptosis,cycle,migration and invasionof ovarian cancer cellsObjective:To study the regulation and mechanism of MNX1-AS1 on ovarian cancer cells.The mechanism of MNX1-AS1 on cell proliferation,cycle,apoptosis and invasion and metastasis of ovarian cancer.Method:the corresponding siRNA short sequences synthesized by the screened LncRNA sequences were transfected in to OVCA433 and skov-3 of human ovarian cancer cell lines;Detect the changes of OVCA433 and skov-3 cell cycles afterMNX1-AS1knowdown by Annexin V/PI double dye flow cytometry.The changes of apoptosis levels of OVCA433 and skov-3 cells after MNX1-AS1 knockdown by Annexin V/PI double dye flow cytometry.Hoechest detects apoptosis of ovarian cancer cells;Transwell detects the invasion and metastasis of ovarian cancer cells.Results:1.Identification of transfection efficiencyThe constructed siRNA and corresponding sequence were transferred to OVCA433 and skov-3 respectively,and the total RNA of each group cells was extracted after transfection of 72h,and the transfection efficiency was tested by QRT-PCR after the reverse transcription into cDNA.The results showed that both in OVCA433 and skov-3 cell lines,siRNA could significantly inhibit the expression level of IncRNA-MNX1-AS1.2.MNX1-AS1 knockout effect on the proliferation and cloning of OVCA433 and skov-3 ovarian cancer cellsCell proliferation and cloning experiments were used.The CCK-8 experimental results showed that the proliferation ability of OVCA433 and skov-3 cells was significantly inhibited after the knockout of MNX1-AX1(P<001).The Edu results showed that the down-regulation of MNX1-AX1 had significantly reduced the proliferation ability of OVCA433 and skov-3 cells(P<0.001).Simulating the clone ability from a single tumor cell to form multiple tumor cells.The smaller and less number of clones formed after OVCA433 and SKOV 3 cells were transfected by siRNA.Statistical analysis shows that MNX1-AS1 down regulation significantly reduced the number of cell clone formation(P<0.001).The results show that the MNX1-AS1 downregulation inhibite the proliferation of OVCA433 and SKOV-3 ovarian cancer cell lines.The reduced clone ability suggests that MNX1-AS1 has played a certain role in promoting clone formation and progress in ovarian cancer.3.MNX1-AS1 knockout effect on ovarian cancer cell cycles of OVCA433 and skov-3 cell lines.Detecting the changes of cell cycle and cycle proportion after MNX1-AS1 knock out in OVCA433 and SKOV-3 cell lines by Using flow cytometry,MNX1-AS1 knockout group significantly increased the percentage of cells in GO/G1 phase and reduced the cell percentage of S phase in OVAC433 and SKOV-3 cell lines.Check.the changes of cell cycle protein expression in downstream by using WesternBlotting.The expression of CDK4 and Cyclin D1 in OVAC3 and skov-3 cells significantly reduced in MNX1-AS1 knockout group after siRNA transfection(P<0.01).Results show that the MNX1-AS1 controlling the changes of proliferation and clone ability of ovarian cancer cell is through participation in the cell cycle to adjust,affect the CDK4 and Cyclin D1 expression changes to affect ovarian cancer cell cycle progression.4.The apoptosis effects on ovarian cancer cells OVCA433 and skov-3 after MNX1-AS1 knock out.detecting the effect on apoptosis of ovarian cancer cells after MNX1-AS1 knockout by using Annexin V/PI double dye flow cytometry detection method.Early apoptotic cells(Annexin V+/pi-)and late apoptotic cells(Annexin V+/PI+)were calculated as the total apoptotic percentage of cells in the four-quadrant diagram.The results showed that the total apoptosis percentage of both ovarian cancer cell lines OVCA433(P<0.01)and SKOV-3(P<0.01)was significantly higher than that in the control group and the negative control group after the knockout of MNX1-AS1.The proportion of the living cells(Anexin V-/PI-)both in OVCA433 MNX1-AS1 knockout groupe and SKOV-3 MNX1-AS1 knockout groupe was significantly lower than that in the control group and the negative control group(P<0.01).Hoechst 33258 was used and observed under a fluorescence microscope.It was found that the apoptosis level of OVAC3 and SKOV-3 cells in MNX1-AS1 knockout group was significantly higher than that in the control group,and the difference was significant(P<0.01).The expression of immune imprinting indicated that the expression of apoptotic protein bax was significantly up-regulated,while the expression of anti-apoptotic protein bcl-2 was significantly decreased(P<0.01).It is indicated that MNX1-AS1 is one of the necessary conditions for anti-apoptosis of ovarian cancer cells,and may play a role in through working with bax and bcl-2 genes.5 Effects on the invasion ability of OVCA433 and skov-3 after MNX1-AS1 knockoutThe wound healing ability of cells after knockout MNX1--AS1 by Using scratch test examines showed that MNX1-AS1 downregulation significantly weaken OVCA433 and SKOV-3(P<0.01)cell lines(P<0.01).Transwell migration experiment of OVCA433 and SKOV-3 with siRNA transfection shows that OVCA433 and SKOV 3(P<0.05)cell movements ability and invasion are inhibited in MNX1-AS1 knockout groups(P<0.05).and there was no significant difference in the control group and the negative control group(P<0.05).Conclusion:1.Knockdown MNX1-AS1 inhibited the proliferation of ovarian cancer cells.2.Knockdown MNX1-AS1 induces cell cycle block and apoptosis of ovarian cancer cell G0/G1 phase.and possibly regulating the cycle by down-regulating the expression levels of CDK4 and CyclinDl,and promoting apoptosis by affecting the expression of Bax and bcl-2.3.Knockdown MNX1-AS1 inhibites the ability of ovarian cancer cell migration,indicates that MNX1-AS1 is an important impact factor in occurring and developmental process of ovarian cancer.and provide evidence for the relevant IncRNA in clinical cancer diagnosis,target therapy of medicine and other fields.Part 3 MNX1-AS1 Effects on tumor groth in mouse model of ovarian cancer xenograftsObjective:Subcutaneous injection of tumor cells was used to construct mouse ovarian cancer xenografts.detect the effect of MNX1-AS1 expression level on tumor growth after injecting MNX1-AS1siRNA into tumor tissues.Provide laboratory basis for the study of biological treatment and immunology of ovarian cancer.Method:1 Prepare the three groups of cell SAP(MNX1-AS1 siRNA group,and NC group and blank control group)corresponding to OVCA433 and SKOV 3 cells respectively;The OVCA433 and skov-3 cells were obtained from the logarithm period,and the culture medium was suspended.Inoculate the cells in the new 10cm diameter cell culture dish until the cell grows to 70%;2 Three groups of cell sap in two cells were injeced in to mice.each group was tested with 3 mice,which all of then formed subcutaneous tumor and Nude mice were executed with a short neck after 4 weeks,The tumor samples were taken to observe the growth size and weighing records;Take moderate amount of tumor sample tissues to detect The expression level of MNX1-AS1 by PCR.3 All quantitative experiments were repeated three times.Data in this study were analyzed with SPSS18 and expressed as mean ± standard deviation(SD).The statistical analysis was considered to be significant when p-value<0.05.Results:1 MNX1-AS1 knockout group after transfection of siRNA significantly reduce the volume and weight of tumor tissus in mice.Compared with NC or control group,the time-dependent analysis showed that the volume and weight of the tumors was significantly suppressed in mice inoculated with MNX1-AS1 siRNA transfected(P<0.05).2 siRNA can specifically inhibit the expression of MNX1-AS1 and inhibit tumor formation in mice.the expression of MNX1-AS1 was significantly reduced(P<0.01)in MNX1-AS1 siRNA tumor tissues(P<0.01)conclusion1.MNX1-AS1 gene knockout significantly inhibited tumor cell growth in mice。2.The results can provide valuable experimental animal models and clinical diagnostic targets for the study of ovarian cancer gene level...