Radiation Induced MiR-1290 Promote Epithelial- Mesenchymal Transition Of Cervical Cancer HeLa Cells

Radiotherapy is an effective and common therapeutic tool for cervical cancer treatment.Clinical data show that the 5-year survival rate of patients with IA cervical cancer after radiotherapy can reach more than 90%.However,cervical cancer patients who experience metastasis or recurrence after radiotherapy are still considered to be solved.The concern is IR paradoxically also promotes tumour recurrence and metastasis.Therefore,understanding the molecular mechanisms of IR-induced tumour invasion and metastasis is required to improve the efficacy of radiotherapy.Invasion and metastasis of tomour are regulated by a network of signaling pathways that involve components and their associated signaling proteins.Epithelial-mesenchymal transition?EMT?is closely linked to the induction of metastasis.Our previous studies show that ⁶⁰Co?-ray radiation induced EMT of multiple tumor cells such as rectal cancer,breast cancer and lung cancer.More and more researches suggested that IR activate EMT-inducing transcription factors,includingHIF-1,Snail,ZEB1 and TGF-?.In recent years,as an important factor in gene expression,transcription and regulation,micro-RNA plays an important role in the development of tumor.microRNA involved in many of the cellular regulatory processes associated with tumor radiation stress responses,including DNA damage repair,cell apoptosis,proliferation and angiogenesis.our previous studies found that in radioresistant cervical cancer Hela cells miR-1290 was significantly increased.Here,this study aims to determine whether miR-1290 is involved in the regulation of invasion and metastasis of cervical cancer cells.We simulate the fractionated therapy of clinical tumor radiotherapy and establish a cervical carcinoma Hela cell line with radioresistance.By using lentivirus vector,we successfully modulate the expression of miR-1290 in Hela cells.In vitro experiment show that radiation-induced miR-1290 promote EMT of cervical cancer HeLa cells.The target gene of miR-1290 was preliminarily analyzed by using the Western blot experiment and the double fluorescent enzyme report gene assay.This study explored the role and mechanisms of recurrence and metastasis of cervical cancer after radiotherapy from the angle of microRNA.The relationship between miR-1290 and cervical cancer recurrence and metastasis can be used as the adjuvant therapy target to improve the curative effect of radiotherapy.Aims:1.To observe radiation induced miR-1290 expression in HeLa cells.2.To analysis the effect of miR-1290 on EMT in HeLa cells.3.To clarify the mechanisms of miR-1290 induced EMT in HeLa cells.Methods:1.qRT-PCR was used to detect the expression of miR-1290 in HeLa cells at different time after different dose radiation of ⁶⁰Co?-ray.2.To simulate the fractionated radiotherapy of clinical treament,the radioresistant HeLa cell variants were established by repeated selection with ⁶⁰Co?-ray and were identified by clonogenic assay.Using qTR-PCR to compare the expression of miR-1290 in radioresistant cells and parental HeLa cells.3.Lentiviral vectors which over-express miR-1290 were established to infect Hela cells.After selection with puromycin,HeLa cells with stable modulation of miR-1290were identified by qRT-PCR.4.Morphological changes of HeLa cells with stable modulation of miR-1290 were observed through a microscope.5.As in vitro experimental model,HeLa cells with stable modulation of miR-1290were used to detect E-cadherin and N-cadherin by Western blot and immunofluorescence staining.6.HeLa cells with stable modulation of miR-1290 and parent HeLa cells were injected subcutaneously into nude mouse to establish model of transplanted tumor.The tumors were resected and sliced into paraffin sections to detect the expression of E-cadherin and N-cadherin by immunohistochemistry staining.7.Scratch assay and transwell chamber assay were applied to observe migration and invasion ability in HeLa cells with stable modulation of miR-1290..8.By using the online bioinformatics software combined with the function of candidate target genes to predict the target genes of miR-1290,which were verified by dual-luciferase reporter gene assay.9.The expression of Smad4 and GSK-3?were determined by Western blot and immunofluorescence staining in HeLa,HeLa-virus-control,HeLa-miR-1290 and HeLa-miR-1290-inhibiton cells.10.Immunohistochemistry staining were used to detect the expression of Smad4 and GSK-3?in paraffin sections mentioned above?Method 6?.11.DNA recombinant technology were choosed to transfect Smad4-siRNA or GSK-3?-siRNA into HeLa cells.Expression of E-cadherin and N-cadherin were determined by Western blot.Results:1.After exposure HeLa cells to single different doses of ⁶⁰Co?-rays,qRT–PCR showed that the expression of miR-1290 in HeLa cells was up-regulated.HeLa-R11 was obtained from HeLa cells that were irradiated repeatedly 11 times.Cloning format assay showed that the radioresistance of HeLa-R11 clones were significantly enhanced upon irradiation,suggesting that radioresistant HeLa cell lines were successfully constructed.qRT–PCR confirmed that expression of miR-1290 in HeLa-R11 were significantly up-regulated.The above results indentified that radiation induced miR-1290 in HeLa cells.2.qRT–PCR confirmed that the HeLa cells with stable modulation of miR-1290?HeLa-virus-control,HeLa-miR-1290 and HeLa-miR-1290-inhibition?were establish successfully.Obvious mesenchymal-like morphological changes?gap between cells widened,end of the cell is elongated,cell polarity disappeared in the form of spindle cells.?were observed in HeLa-miR-1290 cells.3.Western blot confirmed that the expression of E-cadherin was decreased and N-cadherin was increased in HeLa-miR-1290 cells.The immunohistochemistry staining revealed that miR-1290 was positively correlated with the expression of N-cadherin and negatively with E-cadherin in paraffin sections of nude mouse transplanted tumor.4.Scratch assay and transwell chamber assay proved that up-regulating miR-1290increased the ability of migration and invasion;while,down-regulating miR-1290decreased the ability of migration and invasion in HeLa cells..5.The candidate target gene of miR-1290 were predicted by online database.Dual-luciferase reporter gene assay showed that SMAD4 and GSK3B were the target genes of miR-1290.6.Western blot confirmed that the expression of Smad4 and GSK-3?was decreased in HeLa-miR-1290 cells.The immunohistochemistry staining revealed that miR-1290 was positively correlated with the expression of Smad4 and GSK-3?.The above results showed that miR-1290 could inhibit thethe expression of Smad4 and GSK-3?.7.Western blot showed that down-regulating Smad4 in Hela cell reduced the expression of E-cadherin and arised the expression of N-cadherin;down-regulating GSK-3?increased the expression of N-cadherin.The results above indentified that Smad4and GSK-3?inhibited EMT in HeLa cells.Conclusions:1.miR-1290 was increased upon iaradiation in HeLa cells.2.Over-expression of miR-1290 enhanced EMT in HeLa cells.3.SMAD4 and GSK3B were target genes of miR-1290.4.miR-1290-regulated expression of Smad4 and GSK-3?are associated with radiation-induced EMT in HeLa cells.