Study On The Mechanism Of MALAT1-SP1 Transcriptional Feedback Loop In Lung Cancer

Aims:Metastasis associated lung adenocarcinoma transcript 1(MALAT1)is a long non-coding RNA(IncRNA)in nuclear speckles,which is about 8 kb in length and does not encode a protein.MALAT1 is over-expressed in a variety of tumors and is a widely recognized biomarker for tumor diagnosis,staging,and prognosis.So far,many literatures have reported that MALATI plays a very important role in the migration and invasion of many tumor cells.However,the specific mechanism of MALAT1 in tumors is still not very clear.Previous studies on the MALAT1 transcription in our lab showed that SP1(Specific protein 1)is an important transcription factor that regulates MALAT1 gene’s transcription in lung cancer.In addition,down-regulation of SP1 inhibits expression of MALAT1,thereby inhibiting the tumor.We also found that overexpression of MALAT1 increased the expression of SP1 by RT-PCR and western experiments.Thus,a transcriptional loop is formed between the SP1 protein and MALAT1 RNA in lung cancer cells.In this study,two lung cancer cell lines were used as models to elucidate the mechanism of MALAT1-SP1 transcriptional feedback loop in lung cancer.There are three questions that need to be explored.How does MALAT1 contribute to the increase of SP1’s expression?How does this loop affect the growth and metastasis of lung cancer?What is the significance of this loop for the clinical treatment of lung cancer?Methods:Ⅰ.To study the effect of MALATlon SP1’s expression in lung cancer cells:While MALAT1 was over-expressed or knocked-down in lung cancer cells,the expression level of SP1 as well as downstream target genes of SP1 were detected by RT-PCR and Western Blot.The SP1 downstream target genes’ expression level can indirectly reflect the effect of MALATl’s expression on the expression of SP1.Ⅱ.To study the mechanism of the MALAT1 RNA regulating SPl’s expression in lung cancer cells:Cell immunofluorescence assay was used to investigate SP1’s expression and localization while MALAT1 was overexpressed or downregulated.Half-life assay was used to investigate whether the expression of MALAT1 affects the stability of SP1 protein.Immunoprecipitation and UV cross linking were used to investigate whether MALAT1 RNA protects SP1 protein by binding to SP1 protein.Ⅲ.To study the effect of MALATl’s expression on the growth and metastasis of lung cancer in vitro and in vivo:RNA interference was used to knock down MALAT1’s expression in lung cancer cells.The effects of MALAT1 on the migration and invasion of lung cancer cells were investigated by wound healing experiment and transwell assay.In order to investigate if malatl expression was positively correlated to SP1 expression in vivo,A549 cells were injected subcutaneously into the dorsal flanks of mice.Tumor volume was monitored every 3 days and calculated.After four times RNAi expression plasmids injections to the tumor,tumors were isolated,RT-PCR and Western blot analysis were performed to evaluate Spl and MALAT1 expression level.Ⅳ.To study the correlation between MALAT1 and SP1 level in clinical lung cancer samples:RNA and protein were extracted from lung cancer sample and normal lung sample.The correlation between MALAT1 and SP1 was confirmed by RT-PCR and western blot.Results:The results of RT-PCR and western blot showed that overexpressing RNA fragments 1905-3235 of MALAT1 in lung cancer cells could promote the expression of SP1 and SP1’s downstream target genes.Through immunofluorescence experiments,we found that in lung cancer cells overexpressing MALAT1 caused the increasement of SP1 both in nuclei and cytoplasm.By the co-immunoprecipitation and UV cross linking,we found that there was a direct interaction between the transcriptional product RNA of MALAT1 in lung cancer cells and its key regulatory transcription factor SP1 protein,and this combination can stabilize the structure of SP1 protein and prolong its half-life.The results of wound-healing and transwell showed that the up-regulation of MALAT1 expression could promote the migration and invasion of tumor cells in vitro,and the down-regulation of MALAT1 expression had a good inhibitory effect on the migration and invasion of tumor cells in vitro.Furthermore,downregulation of MALAT1 or Spl expression by RNAi also reduces A549 lung cancer cell growth in vivo.In addition,clinical studies have shown that the expression of MALAT1 and SP1 in lung cancer sample were elevated compared to normal lung sample.Conclusion:Ⅰ.There is a direct interaction between the transcriptional product of MALAT1 and its key transcription factor SP1 protein in lung cancer cells,which can stabilize the structure of SP1 protein and prolong its half-life.Thus,the expression level of SP1 is regulated by MALAT1 RNA,and a transcription feedback loop is formed between SP1 and MALAT1.In.Interference with MALATI’s expression has a very good inhibitory effect on tumor growth and metastasis.Ⅲ.There is also a correlation between MALAT1 and SP1 in clinical specimens.Thus,the clarification of this loop providestherapy targets for the clinical treatment of lung cancer.