And Invasion Of Ovarian Cancer Associated 1ncRNA The Initial Screening And MALAT1 Ovarian Cancer Cell Behavior

BackgroundOvarian cancer is the most lethal gynecological malignancy, and lack of techniques for early diagnosis and long-term effective treatment options accounts for the high case fatality rate. Therefore, looking for new biomarkers and therapeutic targets has been a major research focus for many years.lncRNA is an RNA molecule that is longer than200nucleotides and that is not translated into a protein. Although our current understanding of the role of lncRNAs is limited, increasing reports have shown that lncRNAs can regulate gene expression at various levels, including chromatin modification, transcription, and posttranscriptional processing. It has been reported that lncRNAs control various cellular processes, such as proliferation, apoptosis, and invasion, and are implicated in human diseases, including cancer. These days, microarray-based profiling and deep sequencing play an important role in lncRNA research.MALAT1is a lncRNA of about8,000nt and is widely expressed in normal human tissues. In2003, Ji et al. reported for the first time that MALAT1was upregulated in non-small-cell lung cancer and was a prognostic parameter for patient survival. In addition to its potential involvement in lung cancer, MALAT1was also found to be deregulated in hepatocellular carcinoma, cervical cancer, bladder cancer and gallbladder cancer. However, the role of MALAT1in ovarian cancer remains unkown.This paper consists of two parts. In the first part, we presented the lncRNA expression profiles in paired ovarian cancer cell lines with different invasive potentials and found that lncRNAs were differentially expressed in ovarian cancer cells with varying invasive potentials. In the second part, we investigated the effects of MALAT1downregulation on ovarian cancer cells and found that MALAT1downregulation significantly suppressed cell proliferation and metastasis. Besides, downregulation of MALAT1altered the expression of a number of genes associated with cell proliferation, metastasis and/or apoptosis.1. Identification of differentially expressed long noncoding RNAs in human ovarian cancer cells with different invasive potentials ObjectiveTo identify differentially expressed lncRNAs in human ovarian cancer cells with different invasive potentials.MethodsAn in vitro invasion assay was performed to validate the invasive capability of SKOV3and SKOV3.ip1cell lines. Total RNA was then extracted, and microarray analysis was performed. Moreover, nine lncRNAs were selected for validation using qRT-PCR.ResultsCompared with the SKOV3cells, the SKOV3.ip1cells significantly improved in the in vitro invasive activity. Of the4,956lncRNAs detected in the microarray,583and578lncRNAs were upregulated and downregulated, respectively, in SKOV3.ip1cells, compared with the parental SKOV3cells. Seven of the analyzed lncRNAs (MALAT1, H19, UCA1, CCAT1, LOC645249, LOC100128881, and LOC100292680) confirmed the deregulation found by microarray analysis.ConclusionlncRNAs were differentially expressed in ovarian cancer cells with varying invasive potentials. This result indicates that some lncRNAs might exert a partial or key role in epithelial ovarian cancer metastasis. Further studies should be conducted to determine the roles of these lncRNAs in ovarian cancer metastasis.2. Downregulation of the long noncoding RNA MALAT1suppressed proliferation and metastasis in ovarian cancer cellsObjectivesTo investigate the effects of MALAT1downregulation on proliferation and metastasis in ovarian cancer cells.Methods 1. qRT-PCR was used to evaluate the expression levels of MALAT1in human ovarian cancer cell line SKOV3and human ovarian surface epithelial cells HOSE.2. Establishment of stable MALAT1-knockdown cells (SKOV3-MALAT1-KD) and control cells (SKOV3-NC).3. Cell proliferation assay and clony formation assay were done to evaluate the effect of MALAT1downregulation on cell proliferation in vitro; Cell migration assay and invasion assay were used to assess the effects of MALAT1downregulation on cell migration and invasion in vitro; Apoptosis was detected by flow cytometry.4. For in vivo tumorigenic assay, SKOV3-MALAT1-KD and SKOV3-NC cells were injected subcutaneuosly into nude mice. Once palpable tumors developed, caliper measurements were taken every three days and tumor volume was calculated.5. Total RNA was extracted from SKOV3-MALAT1-KD cells and the control cells, and microarray analysis was performed. Moreover,20differentially expressed genes which have been reported to be associated with cell proliferation, metastasis and/or apoptosis were selected for validation using qRT-PCR.Results1. qRT-PCR showed that MALAT1was significantly upregulated in ovarian cancer cell line SKOV3compared to human ovarian surface epithelial cells HOSE.2. Stable MALAT1-knockdown cells (SKOV3-MALAT1-KD) and control cells (SKOV3-NC) were successfully established.3. MALAT1downregulation significantly suppressed proliferation, migration and invasion in vitro (P<0.01); Compared with the control group, SKOV3-MALAT1-KD cells showed an increased level of apoptosis (P<0.01).4. The results of in vivo tumorigenic assay showed that the average tumor volume of the SKOV3-MALAT1-KD group was significantly smaller than that of the control group (P<0.01).5. Compared with the control cells,921genes in the SKOV3-MALAT1-KD cells were deregulated at least two-fold; qRT-PCR results showed that19of these genes confirmed the deregulation found by microarray analysis. ConclusionsMALAT1downregulation significantly suppressed proliferation and metastasis in ovarian cancer cells. Moreover, MALAT1downregulation inhibited tumor growth in vivo. Downregulation of MALAT1altered the expression of a number of genes associated with cell proliferation, metastasis and/or apoptosis. Taken together, our findings define a major oncogenic role for MALAT1, which may offer an attractive new target for therapeutic intervention in ovarian cancer.