The Functional Role And Mechanism Of Mitochondrial Leucyl-tRNA Synthetase Deregulation In Clear Cell Renal Cell Cancer

Renal cell cancer(RCC)is a kind of malignant lesion which originates from renal tubular epithelial cells,80?85%of RCC cases are clear cell RCC(ccRCC).Nowadays,the molecular mechanisms in occurrence,progression and metastasis of RCC have not been fully recognized yet,so further research is desiderated in this area.Previously,our team had detected the gene profile of ccRCC tissues and adjacent kidney tissues by whole genome sequencing technique,and differential genes between the two groups were identified.Deletion or mutation of mitochondrial leucyl-tRNA synthetase(LARS2)gene were frequently found in ccRCC tissues,leading to afunction of LARS2 protein.So it suggested that LARS2 may play an important role in the occurrence and progression of ccRCC.Objective:To study the expression levels and biological effects of LARS2 in ccRCC,and to explore the mechanisms of its action.Materials and Methods:(1)The qRT-PCR,Western blot,and ICC methods were used to detect the RNA and protein expression levels of LARS 2 in ccRCC cell lines and renal tubular epithelial cell lines.Then qRT-PCR,Western blot and IHC experiments were performed to detect the RNA and protein expression levels of LARS2 in ccRCC tissues and paraneoplastic kidney tissues.In addition,we explored the relationship between LARS2 expression levels and clinicopathological characteristics of ccRCC patients,and analyzed the influence of LARS2 on prognosis of ccRCC patients.(2)The LARS2 over-expression cells were established in ccRCC cell lines OS-RC-2,Caki-1 and renal tubular epithelial cell line HKC,besides control cells were also established.The LARS2 low-expression and control cells were also established in HKC cell line.Then the level of proliferation,clone formation ability,cell cycle,level of apoptosis,ATP content,level of reactive oxygen species(ROS)were detected in these cells,and OS-RC-2 cells were used to form neoplasms in kidney of nude mice.The results between stable transfection cells and control cells were compared.(3)Western blot was used to detect activation level of Ras/Raf/MEK/ERK,AMPK?,HIF-1?/mTOR,and VHL/HIF-2? pathways in OS-RC-2,Caki-1,and HKC cells with LARS2 over-expression or low-expression.Then the activation levels between stable transfection cells and control cells were compared.Results:(1)Compared with renal tubular epithelial cells,the mRNA and protein expression levels of LARS2 were reduced in ccRCC cells.Compared with paired paraneoplastic kidney tissues,the mRNA and protein expression levels of LARS2 were decreased significantly in ccRCC tissues(P<0.05).The mRNA levels of LARS2 were correlated with clinicopathological characteristics of ccRCC patients and could affect the prognosis of ccRCC patients,and patients with LARS2 low-expression generally had a poorer prognosis(P<0.05).(2)Compared with control cells,the LARS2 over-expression cells proliferated slower,formed less clones,suffered more G1/S checkpoint retardation,had more cell apoptosis,higher cellular ATP content,lower ROS level,and formed smaller renal neoplasms in kidney of nude mice(all P<0.05).Compared with control cells,the LARS2 low-expression cells proliferated faster,formed more clones,had lower ATP content and higher ROS level(all P<0.05).(3)Compared with control cells,the LARS2 over-expression cells showed lower activation level of Ras/Raf/MEK/Erk pathway,AMPK? pathway,and mTOR pathway(all P<0.05),while the activity of VHL/HIF-2? pathway remained unchanged(P>0.05).Compared with control cells,the LARS2 low-expression cells showed higher activation level of Ras/Raf/MEK/ERK pathway,AMPKa pathway,and mTOR pathway(all P<0.05),while the activity of VHL/HIF-2a pathway remained unchanged(P>0.05).Conclusions:This study found that the mRNA and protein expression levels of LARS2 were decreased significantly in ccRCC tissues and cell lines.LARS2 low-expression was a risk factor for the prognosis of ccRCC patients.Knockdown LARS2 could favor the progression of ccRCC cells,as it activated Ras/Raf/MEK/ERK pathway,AMPKa pathway,and mTOR pathway,while had no influence on the activation level of VHL/HIF-2a pathway.