| ObjectiveHepatocellular carcinoma(HCC)is still a major health burden in China considering its high incidence and mortality.Long non-coding RNAs(lnc RNAs)were found playing vital roles in tumor progression,suggesting a new way of diagnosis and prognosis prediction,or treatment of HCC.This study was designed to investigate the role of HIF1A-AS1 during the progression of HCC,and to explore its related mechanisms.Methods1.We first detected the expression of HIF1A-AS1 in 50 pairs of HCC specimens and corresponding adjacent normal tissues by q RT-PCR.Additionally,we analyzed the correlation between HIF1A-AS1 expression and clinicopathological characteristics including age,sex,tumor size,TNM stage,lymph node metastasis,recurrence rate and survival time in HCC patients,which were divided into high HIF1A-AS1 group(n=25)and low HIF1A-AS1 group(n=25)with the median value of HIF1A-AS1 expression as a cutoff point.2.We detected HIF1A-AS1 expression levels in two HCC cells(7721、Huh7)and the normal hepatocyte cell line L02 under different culture conditions.Furthermore,we analyzed the relationship between HIF1A-AS1 expression and the starvation time(0h、12h、24h、48h、72h)in 2 HCC cell lines(7721、Huh7).3.Si RNAs targeting HIF1A-AS1(si-HIF1A-AS1)and negative control(si-NC)were synthesized and transfected into HCC cells respectively.The inhibition efficiency of si-HIF1A-AS1 was verified by q RT-PCR assay.We examined the effects of inhibiting the expression of HIF1AAS1 on the proliferation and apoptosis of hepatocellular carcinoma cells in a starvation culture state using the CCK8 and flow cytometry.4.We explored the changes in the expression of autophagy-related molecules under starvation condition,the relationship between HIF1A-AS1 expression and cell autophagy,and possible downstream mechanisms by western blot assay.Results1.HIF1A-AS1 expression was obviously up-regulated in HCC specimens when compared with that in matched normal tissues.Statistical analysis revealed that high level of HIF1A-AS1 was significantly correlated with tumor size,TNM stage and lymph node metastasis.There was no significant correlation between HIF1A-AS1 expression and other clinicopathological features including age,gender.Furthermore,patients in the high HIF1A-AS1 group had a shorter overall survival and a worse disease-free survival than low HIF1A-AS1 group.2.On normal culture condition,the expression level of HIF1A-AS1 was significantly higher in HCC cell lines(7721 and Huh7)than that in normal hepatocyte cell line L02.Under starvation condition,HIF1A-AS1 expression was significantly increased in both HCC and normal hepatocyte cell lines.Furthermore,the expression of HIF1A-AS1 was increased with the prolongation of starvation culture in both 7721 and Huh7 cell lines.3.The result from CCK-8 assay showed that the viability of HCC cells transfected with siHIF1A-AS1 was significantly lower than that of si-NC group under starvation condition.Flow cytometry assay confirmed that inhibition of HIF1A-AS1 could enhance starvation-induced HCC cells apoptosis.4.The expression of autophagic marker(BECN1 and LC3)was significantly increased in 7721 and Huh7 cell lines after 12 h starvation culture.After 12 h starvation-induced,the increased expression of BECN1 and the conversion of LC3 I to LC3II(LC3 II/I)was significantly reduced in both 7721 and Huh7 cell lines transfected with si-HIF1A-AS1.Furthermore,the expression of HIF-1α and phosphorylated m TOR was significantly reduced in 7721 and Huh7 HCC cell lines after HIF1A-AS1 inhibition.Conclusions1.lnc RNA HIF1A-AS1 expression might be associated with the HCC progression and prognosis.2.lnc RNA HIF1A-AS1 expression might be correlated with nutrient-deficient induced HCC cells biological behaviors.3.lnc RNA HIF1A-AS1 might regulate starvation-induced HCC cells autophagy through HIF-1α/m TOR pathways to promote the progression of HCC.4.lnc RNA HIF1A-AS1 may be one of the potential targets for the treatment of HCC. |
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