Research On LHX2 Promoting Malignancy In Osteosarcoma And Its Mechanism

Background and purposes:Osteosarcoma(OS)is a common malignant tumor of bone that occurs in children and elderly.The standard treatment for osteosarcoma includes surgical resection of local lesions and the use of chemotherapy drugs before and after the operation.The application of this scheme increases the 5 year survival rate(65%)in osteosarcoma patients.However,the clinical treatment of osteosarcoma has not made significant progress in the last thirty years due to the existence of Tumor heterogeneity.Therefore,It is vital to understand the pathogenesis of osteosarcoma better and look for new targets for the development of osteosarcoma treatment.LIM-homeobox gene 2(LHX2)is upregulated in various cancers and closely related to tumor growth,metastasis and poor prognosis.But whether LHX2 exerts as an oncogene in osteosarcoma remains elusive.The correlation between autophagy and tumor is cloze and complicated,the role of autophagy in tumor progression is a double-edged sword,and autophagy participates in the regulation of proliferation,metastasis,apoptosis and other malignant phenotypes in osteosarcoma.MicroRNA(miRNA)is a single-stranded non-coding RNA composed of 18-25 nucleotides.The mature miRNA inhibits gene expression by binding to the 3' UTR of the target mRNA,therefore play a critical role in cells.Recent studies indicate that miRNA is closely related to tumor progression.Previous research showed that miR-129-5p inhibits the growth and metastasis in osteosarcoma,but its downstream and underlying molecular mechanism is not clear.This study aims to explore the expression,function of LHX2 and its possible molecular mechanism in osteosarcoma.Besides,we explore the mechanism for its aberrant expression from the point of view of miRNA.The completion of this study will provide new strategies and interventions for the prevention and treatment of osteosarcoma.Methods:1.LHX2 expression and its correlation with clinicopathological parameters in osteosarcoma.1)GEO database was employed to analyze the LHX2 expression in 18 paired OS tissues;2)OS tissue microarrays(TMA)were used to analyze the expression and contribution of LHX2 in 40 osteosarcoma patients.3)qRT-PCR and Western blot were used to detect the mRNA and protein expression in normal osteoblast cell line and osteosarcoma cell lines 143 B,MG63,AND Saos-2.4)A total of 71 osteosarcoma tissues were obtained,and LHX2 expression was evaluated by immunohistochemistry.The correlation between LHX2 expression and clinical parameters and prognosis was analyzed.2.The effect of LHX2 on osteosarcoma cells proliferation,migration and invasion in vitro.1)LHX2 silence lentivirus vectors(Lv-shLHX2)were constructed by RNAi technique.Osteosarcoma cell lines 143 B and MG63 were transfected with shLHX2 and control lentivirus vectors.The transfection efficiency was verified by qRT-PCR and Western blot.2)CCK8,colony formation assays were performed to detected cell proliferation ability in LHX2 silence cells.3)Wound healing,transwell migration and invasion assays were performed to investigate the cell migration and invasion ability in LHX2 silence cells.3.The effect of LHX2 on osteosarcoma cells autophagy and its possible mechanism.1)R2 database were employed to investigate the correlation between LHX2 and autophagy-related gene and protein expression.2)qRT-PCR and Western blot were performed to detect the autophagy-related gene and protein expression in LHX2 silence osteosarcoma cells.3)GFP-RFP-LC3 fusion assay was performed to observe the formation of the autophagosome in LHX2 silence cells.4)Expressions and activity of mTOR pathway signaling-related proteins were detected by Western blot.4.Effect of LHX2 on osteosarcoma growth and metastasis in vivo.1)Orthotopic spontaneous metastasis OS animal models were established using 143 B cells stably expressed firefly luciferase and transfected with the shLHX2 and control lentivirus.2)Tumor growth curve was established to observe the growth of osteosarcoma.3)Lung tissues were dissected and the foci of pulmonary metastasis were detected by bioluminescent imaging and HE staining.4)Proliferation marker Ki67 and autophagy marker LC3 B expressions were detected by immunehistochemistry(IHC)in tumors.5.LHX2 is directly targeted by miRNA-129-5p.1)Bioinformatics prediction tool Targetscan was used to screen the miRNA that might target the 3'UTR region of LHX2.LHX2 3' UTR Mutant lentivirus vector was constructed according to the bingding site of miR-129-5p and LHX2.2)Dual-luciferase assay was performed to evaluate whether miR-129-5p could bind to the 3'UTR of LHX2.3)LHX2 protein expression was detected by Western blot after osteosarcoma cells were transfected with miR-129-5p overexpression or inhibition lentivirus vectors.6.miR-129-5p inhibits osteosarcoma cells proliferation,migration and invasion by targeting LHX2 in vitro.1)143B and MG63 cells stably transfected with corresponding lentivirus vectors were divided into three groups: control group,miR-129-5p overexpression group,miR-129-5p plus LHX2 group.2)CCK8,colony formation assays were performed to detected cell proliferation ability.3)Wound healing,transwell migration and invasion assays were performed to investigate the cell migration and invasion ability.7.miR-129-5p inhibits osteosarcoma growth and metastasis by targeting LHX2 in vivo.1)Orthotopic spontaneous metastasis OS animal models were established using 143 B cells stably expressed firefly luciferase and transfected with control,miR-129-5p overexpression,miR-129-5p plus LHX2 lentivirus vectors.2)Tumor growth curve was established to observe the growth of osteosarcoma.3)Lung tissues were dissected and the foci of pulmonary metastasis were detected by bioluminescent imaging and HE staining.Results:1.From 18 primary OS and paired adjacent normal tissues in the GEO database,LHX2 expression was significantly higher in OS tissues(P=0.0069);The immunohistochemistry staning of OS tissue microarrays(TMA)revealed that LHX2 was positively stained in 34 samples(34/40,85%).47%(19/40)of which had an IHC score of(+),33%(13/40)had an IHC score of(++),and 5%(2/40)had an IHC score of(+++);Both the mRNA and protein levels of LHX2 were significantly higher in osteosarcoma cell lines Saos-2,143 B and MG63 compared to normal human osteoblast cell line hFoB 1.19;From 71 OS samples,LHX2 expression was closely correlated with pulmonary metastasis(P=0.013)and Enneking stage(P=0.007).Kaplan-Meier(KM)analysis revealed that high LHX2 levels were associated with poor overall survival(P=0.0052)and metastasis-free survival(P=0.0414)in OS patients.2.Three shLHX2 lentiviruses were successfully transfected into 143 B and MG63 cells.Both the mRNA and protein level of LHX2 were significantly decreased in LHX2 silence group.And shLHX2-3 showed the highest knockdown efficiency.The results of CCK8,colony formation,and Western blot assays revealed that LHX2 silencing significantly decreased proliferation ability and colony numbers;The results of wound healing,transwell migration and invasion assays revealed that LHX2 silencing significantly reduced the migration rates and invasive ability.3.Data from R2 database revealed that LHX2 expression was negatively correlated with autophagy-related genes LC3 and Beclin1.The results of qRT-PCR and Western blot revealed that the mRNA and protein level of autophagy-related genes were significantly increased in LHX2 silence osteosarcoma cells.GFP-RFP-LC3 fusion assay revealed that the number of autophagosomes was increased in LHX2 silence osteosarcoma cells.The result of Western blot revealed that LHX2 silencing did not alter total Akt,mTOR and ULK1 levels,but phosphorylated p-Akt(Ser473)and p-mTOR(Ser2448)levels significantly declined.In addition,the levels of p-ULK1(Ser757)were increased.4.The result of tumor-bearing assay indicated that LHX2 silencing inhibits the growth of local tumor and pulmonary metastasis.The result of immunehistochemistry(IHC)indicated that the protein level of proliferation marker Ki67 was decreased and autophagy marker LC3 B was increased in LHX2 silence group.5.The result of dual-luciferase assay revealed that miR-129-5p directly targets at LHX2 by binding to its 3'UTR;The result of Western blot assay revealed that LHX2 protein level was significantly decreased in cells transfected with miR-129-5p overexpression lentivirus.In contrast,the inhibition of miR-129-5p promoted the protein level of LHX2.6.The results of CCK8,colony formation,and Western blot assays revealed that miR-129-5p suppresses the proliferation and migration of both cell lines,and this effect could be partially rescued by LHX2 overexpression;The results of wound healing,transwell migration and invasion assays revealed that miR-129-5p-mediated migration and invasion inhibition can be partially rescued by LHX2 overexpression.7.The result of tumor-bearing assay indicated that miR-129-5p overexpression inhibits osteosarcoma growth and metastasis,and this effect can be rescued by LHX2 overexpression.Conclusion:LHX2 is upregulated in both osteosarcoma tissues and cell lines,and closely related to pulmonary metastasis and Enneking stage.Higher LHX2 expression predicted poor diagnosis of OS patients.LHX2 silencing inhibits proliferation and metastasis and enhances the autophagy level of osteosarcoma cells.The underlying mechanism may be related to the inactivation of Akt/mTOR/ULK1 signaling.Besides,miR-129-5p inhibits osteosarcoma proliferation and metastasis by targeting LHX2.