Effect Of LncRNA HULC On Proliferation And Invasion In Glioblastoma Through PTEN Methylation And Histone Acetylation

Background and purpose:Glioblastoma multiforme（GBM）is the most common solid tumor that is primarily derived from central nervous system（CNS）with high grade according to WHO classification.Thus,GBM is hard to be completely cured and tends to throw severe impacts on clinical prognosis with extremely low 5-years survival rate.Currently,the treatments of GBM include surgical resection,chemotherapy and radiotherapy.Besides,target therapy is also widely used in clinic.Disappointedly,the curative effect continues to be poor.Long noncoding RNA（Lnc RNA）owns various and complicated functions,in which modulating tumorigenesis and tumor progression are important.With the continuously renewal of researches,Lnc RNA HULC was found exerting tumor promotion function in liver cancer,glioblastoma,pancreatic cancer etc.However,the explicit mechanisms still remain uncovered.Phosphatase and tensin homolog deleted on chromosome ten（PTEN）is an essential tumor suppressor gene in normal cells and inactivation of it can result in canceration of normal cells.On the contrary,when the expression of PTEN is increased,the activity of PI3K/AKT signal pathway can be inhibited.As a result,the ability of tumor proliferation and invasion are enhanced.The regulation of gene expression not only includes the change of DNA sequence,but also includes the influences on epigenetic modification,transcription level and translation level etc.DNA methylation and histone acetylation can regulate gene expression on m RNA level to some extent.Relative drugs have already been used clinically,while its anti-tumor effects on solid tumor still remain controversial.When PTEN is methylated in its promoter region,its expression is reduced.Also,the ability to inhibit PI3K/AKT signal pathway is weakened.Thus,tumor proliferation and invasion are facilitated.In this study,we constructed stable transfected glioblastoma cell line U251 to explore the influences of Lnc RNA HULC on the methylation level in the promoter region of PTEN and histone acetylation to further effect cell proliferation,migration and invasion.Methods:1.Glioblastoma cell line U251 was stably transfected with Lnc RNA HULC,and were divided into Lnc RNA HULC highly-expressed group（HULC group）and negative control group（vec group）;2.The expression level of Lnc RNA HULC and PTEN in each group were tested by q RT-PCR;3.The methylation level in the promoter region of PTEN in each group were detected by BSP;4.The expression level of PTEN,histone H3/H4,ac-histone H3/H4 were measured by WB;5.Cell proliferation,migration and invasion were verified by colony formation assay,CCK8 assay,wound-healing assay and transwell assay;Results:1.The relative expression level of Lnc RNA HULC in HULC group was obviously higher than vec group while PTEN showed the opposite,suggesting that stable transfected cell lines were constructed successfully and HULC can reduce the expression of PTEN on m RNA level;2.The methylation level in the promoter region of PTEN was lower in HULC group then vec group,underlying that the different expression of PTEN on m RNA level in each group maybe correlated with the methylation level in the promoter region induced by HULC and Lnc RNA HULC can reduce the methylation level in the promoter region of PTEN;3.The expression level of PTEN in HULC group was dramatically lower than vec group,indicating that HULC can inhibit the expression of PTEN on protein level.The expression level of histone H3/H4 showed no differences between HULC group and vec group,while ac-histone H3/H4 was significant lower in HULC group then vec group,proving that HULC can decrease the expression level of histone acetylation.4.Colony formation rate,OD values,wound-healing ratio and cell invasion numbers in HULC group were higher than vec group,demonstrating that HULC can promote cell proliferation,migration and invasion in glioblastoma;Conclusion:Lnc RNA HULC reduce the methylation level in the promoter region of PTEN and histone acetylation,inhibit the expression of PTEN,to promote cell proliferation and invasion in GBM cell line U251.