| Lung cancer is the most common malignant tumor,and its morbidity and mortality rank first in the world for many years.Although the traditional treatment methods such as surgery,radiotherapy and chemotherapy have improved in recent years and the overall treatment effect of lung cancer patients has improved,the overall prognosis is still poor.Tumor invasion and metastasis are the most important reasons for poor prognosis.An important intrinsic factor of epithelial tumors to infiltration and metastasis is obtaining mesenchymal cell ability which is epithelial-mesenchymal transition(EMT).EMT could be abnormally activated at any stage of cancer progression and played an important role in the growth,invasion,metastasis and drug resistance of tumors.Long non-coding RNA(Lnc RNA)refers to non-coding RNA whose length is more than 200 nt.In recent years,a large number of studies have shown that many of lnc RNAs are involved in the occurrence,progression and metastasis of lung cancer.Long non-coding RNA 00511(LINC00511)is up-regulated in lung cancer,and the overexpression of LINC00511 is related to invasion and metastasis of tumors,but the mechanism is unclear.The present study gradually elucidates the role of LINC00511 in proliferation,migration and invasion of lung cancer cells and reveals the underlying molecular mechanism through in vitro study.This research is divided into three parts:(1)To explore the effects of LINC00511 in proliferation,migration and epithelial-mesenchymal transition of lung cancer cells and its underlying molecular mechanism.(2)To explore the role of LINC00511 in TGF-β1-induced EMT,invasion and migration of lung cancer cells.(3)To explore the molecular mechanism of LINC00511 in TGF-β1-induced EMT,invasion and migration of lung cancer cells.The results show that:(1)LINC00511 promotes the proliferation,migration and epithelial-mesenchymal transition of lung cancer cells.PTEN is the key intermediate molecule of LINC00511 in regulating the proliferation,migration and epithelial-mesenchymal transition of lung cancer cells.(2)LINC00511 is the key intermediate molecule of TGF-β1 induced promotion of lung cancer.Knockdown of LINC00511 could inhibit the TGF-β1-induced invasion and migration of lung cancer cells via regulating MMPs and EMT.(3)LINC00511 regulates the TGF-β1-induced epithelial-mesenchymal transition,invasion and migration of lung cancer cells through the mi R-183-5p/ZEB2 axis.Part 1.The effects of LINC00511 in proliferation,migration and epithelial-mesenchymal transition of lung cancer cells and the underlying molecular mechanismObjective: The purpose of this part is to explore the effects of LINC00511 on proliferation,migration and epithelial-mesenchymal transition of lung cancer cells and the underlying molecular mechanism through in vitro study.Methods: We first synthesized small interfering RNA(si RNA)of LINC00511 and transfected lung cancer cells with si RNA to silence LINC00511.Then we analyzed the proliferation and migration of lung cancer cells after LINC00511 knockdown.Western Blotting method was used to detect the expressions of EMT-related proteins in lung cancer cells in LINC00511 knockdown group and control group.Meanwhile,the m RNA expression and protein expression of PTEN in LINC00511 knockdown group and control group were analyzed by q RT-PCR and western blotting assays.We further evaluated the effects of LINC00511 overexpression in invasion,metastasis and EMT of lung cancer cells.Rescue experiments were conducted to verify the effect of PETN in LINC00511 regulated invasion,migration and EMT of lung cancer cells.Results: siRNA of LINC00511 downregulated its expression in lung cancer cell lines,and knockdown of LINC00511 significantly inhibited the proliferation and migration of lung cancer cells.The epithelial-mesenchymal transition of lung cancer cells was also suppressed after loss of LINC00511.Moreover,we found that silencing LINC00511 significantly increased the m RNA and protein expressions of PTEN.On the contrary we found that overexpression of LINC00511 promoted cell migration,proliferation and EMT of lung cancer cells and also reducing PTEN expression.Knockdown of PTEN in LINC00511-silenced cells can reverse the proliferation,migration and EMT of lung cancer cells and recover downstream AKT signaling pathway which were inhibited by knockdown of LINC00511.Conclusion: LINC00511 promotes the proliferation,migration and epithelial-mesenchymal transition of lung cancer cells.PTEN is a key regulator in KINC00511 promoted proliferation,migration and epithelial-mesenchymal transition of lung cancer cells.Part II.The role of LINC00511 in the invasion,migration and epithelial-mesenchymal transition of lung cancer cells induced by TGF-βObjective: The purpose of this part was to explored the role of LINC00511 in the TGF-β1-induced invasion,migration and EMT of lung cancer cells.Methods: To study the effect of LINC00511 on lung cancer metastasis induced by TGF-β1,lung cancer cell lines were divided into four groups: control group,TGF-β1 group,TGF-β1+si NC group,TGF-β1+si Linc00511 group.TGF-β1 increased the expression of LINC00511 in lung cancer cells,and LINC00511 si RNA reduced the expression of LINC00511 which was induced by TGF-β1.In order to study the effect of LINC00511 on TGF-β1-induced lung cancer metastasis,we examined the expression levels of vimentin,N-cadherin,E-cadherin and ZEB2 in lung cancer cells among the four groups.In addition,we compared the expression levels of MMP-7 and MMP-13 in the four groups.Finally,we observed changes of invasion and migration ability in lung cancer cells among the four groups.Results: The results showed that TGF-β1 could induce the expression of LINC00511 in lung cancer cells,and si RNA of LINC00511 coulddecrease the LINC00511 expression induced by TGF-β1.TGF-β1 promoted epithelial-mesenchymal transition of lung cancer cells,while knockdown LINC00511 inhibited epithelial-mesenchymal transition induced by TGF-β1.TGF-β1 increased the expression of ZEB2,andknockdown LINC00511 inhibited the TGF-β1 induced ZEB2.TGF-β1 could promote the expressions of MMP-7 and MMP-13 in lung cancer cells,and knockdown LINC00511 inhibited TGF-β1 induced MMPs upregulation.TGF-β1 could promote the invasion and migration of lung cancer cells,knockdown of LINC00511 could inhibit the invasion and migration of lung cancer cells induced by TGF-β1.Conclusion: LINC00511 is a key regulator in the development of lung cancer induced by TGF-β1.Knockdown LINC00511 can inhibit the invasion and migration of lung cancer cells induced by TGF-β1 by regulating MMPs and EMT.Part III.The molecular mechanisms of LINC00511 affecting invasion,migration and EMT induced by TGF-β1 in lung cancer cellsObjective: The purpose of this part was to explored the possible mechanism of LINC00511 in the TGF-β1-induced invasion,migration and EMT of lung cancer cells.Methods: In the second part,we found that knockdown of LINC00511 could decrease the expression of ZEB2,and inhibit the EMT,invasion and migration of lung cancer cells.To further clarify the specific mechanism of ZEB2 regulated by LINC00511 in the process of invasion and metastasis induced by TGF-β1,we used pc DNA3.1 vector to up-regulate the expression of ZEB2 after knockdown LINC00511 in lung cancer cell lines induced by TGF-β1,then the epithelial-mesenchymal transition,invasion and migration of lung cancer cells were observed.Considering the general phenomenon that Lnc RNA can competely bind to micro RNA and promote cancer progression,we use Star Base v2.0 database to predict that LINC00511 has potential binding sites with anti-cancer mi R-183-5p,and meanwile we predict that mi R-183-5p can bind to ZEB2 3’UTR in lung cancer cells.Therefore,we hypothesize that LINC00511 can competely bind to mi R-183-5p and promote EMT in lung cancer cells.To verify this hypothesis,we synthesized LINC00511-mut plasmid to interfere with the interaction between LINC00511 and mi R-183-5p.We compared the expression of ZEB2 among control group,mi R-183-5p group,mi R-183-5p and Linc00511 group and mi R-183-5p and Linc00511 mut group.In addition,we also examined the differences in the expressions of epithelial-mesenchymal transition-related molecules in lung cancer cells among the four groups after adding TGF-β1,further clarifying the possibility that LINC00511 can promote EMT of lung cancer cells by competitive binding to mi R-183-5p.Results: The results showed that overexpression of ZEB2 could reverse the inhibition of si Linc00511 on TGF-β1-induced epithelial-mesenchymal transition,as well as the migration and invasion of lung cancer cells induced by TGF-β1.LINC00511 regulated the expression of ZEB2 through competitive binding of mi R-183-5p,and LINC00511 regulated the epithelial-mesenchymal transition of lung cancer cells induced by TGF-β1 via mi R-183-5p/ZEB2 axis.Conclusion:LINC00511 regulates the epithelial-mesenchymal transition,invasion and metastasis of lung cancer cells induced by TGF-β1 via regulating mi R-183-5p/ZEB2 axis. |
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