LncRNA-HOTTIP Regulates Renal Cell Carcinoma Progression And The Involved Molecular Mechanism Research

Long non-coding RNAs(lnc RNAs)are a class of RNA molecules whose length are more than 200 nucleotides,without possessing the coding potential.Currently,lnc RNAs have become a key regulator in various cellular processes.They can promote or inhibit gene expression during physiological and pathological conditions.An increasing number of researches show that lnc RNAs act as oncogenes or tumor suppressors in cancers.HOTTIP,which is located at the 5’ end of the HOXA locus,is a newly discovered lnc RNA in recent years.Lnc RNA-HOTTIP was reported to promote the progression of several cancers.Furthermore,autophagy is one kind of cellular homeostasis mechanism.It plays an important role in cell survival,growth and development.Autophagy maintains at the basal level in un-stimulated cells under normal conditions,while it is enhanced to act as the source of internal energy in the absence of nutrients or under stressing.Moreover,autophagy can also be enhanced when the tumor cells are stimulated,protecting the tumor cells or promoting their death.That is to say,autophagy plays a dual role in promoting or inhibiting cancer.It is well known that renal cell carcinoma(RCC)is a common malignant tumor in the urinary system,meanwhile,it is also an important member in cancers family.The morbidity and mortality of RCC have not been improved because of the limitations of its diagnosis and treatment,posing a big threat to human health and life.Therefore,it is very urgent to find a more sensitive diagnostic marker and therapeutic target.Recently,studies found that lnc RNAs play an important role in the regulation of autophagy.So based on the emerging knowledge of lnc RNA-autophagy-cancer axis,we explored the relationship between lnc RNA-HOTTIP and RCC autophagy as well as RCC progression,hoping to find a new target for RCC diagnosis and treatment.Part 1 The expression and roles of lnc RNA-HOTTIP in RCCObjective To explore the expression of HOTTIP in RCC,and to assess the effects of HOTTIP expression levels on RCC progression and patients prognosis.Methods In this research,we firstly measured the expression of HOTTIP in 42 pairs of RCC tissues and their adjacent normal kidney tissues,and followed up the 42 patients for 40 months.Meanwhile,we analyzed the correlation between the clinicopathological features of these patients and their HOTTIP expression levels,and assessed the prognosis of these patients.Moreover,we also measured the expression of HOTTIP in 4 RCC cell lines and 1 normal renal tubular epithelial cell line.Subsequently,we selected 2 suitable RCC cell lines to silence HOTTIP and 1 suitable RCC cell line to over-express HOTTIP,and observed the effects of HOTTIP expression levels on RCC cells proliferation,migration and invasion.Finally,we constructed a nude mouse xenograft model and a nude mouse tail vein injection model using the stably transfected RCC cell lines,and observed the effects of HOTTIP expression levels on tumors growth and metastasis in vivo.Results The results showed that the expression of HOTTIP was significantly up-regulated in RCC tissues compared with the adjacent normal kidney tissues.Moreover,the expression level of HOTTIP was closely related to the TNM stage,histological grade and lymph node metastasis of RCC,except for the gender,age and tumor size of patients.In addition,the expression level of HOTTIP was negatively correlated with the patients prognosis.Similarly,the expression of HOTTIP in RCC cells was also significantly up-regulated compared to the normal renal tubular epithelial cell.The results in vitro showed that HOTTIP silencing significantly inhibited RCC cells proliferation,migration and invasion;HOTTIP over-expression significantly promoted RCC cells proliferation,migration and invasion.The results in vivo further indicated that HOTTIP silencing significantly inhibited the tumors growth and metastasis;HOTTIP over-expression significantly promoted the tumors growth and metastasis.Conclusions Lnc RNA-HOTTIP is highly expressed in RCC and acts as an oncogene.Its expression level can predict the prognosis of RCC patients.Part 2 The relationship between lnc RNA-HOTTIP and autophagy in RCCObjective To evaluate the relationship between HOTTIP and autophagy in RCC,and to further explore the way of HOTTIP expression affecting RCC progression.Methods In this research,we firstly treated the stably transfected RCC cell lines constructed above with 3-MA(a common autophagy inhibitor)and RAPA(a common autophagy inducer).Then we observed the effects of HOTTIP expression levels and the drugs treatment on these RCC cells autophagy.In addition,we also measured the autophagy of tumor tissues in the nude mouse xenograft model constructed above.Finally,in the case of adding 3-MA and RAPA,we examined again the effects of HOTTIP expression levels on RCC cells proliferation,migration and invasion.Results The results showed that HOTTIP silencing significantly induced the autophagy in RCC cells and tumor tissues,and HOTTIP over-expression significantly inhibited the autophagy in RCC cells and tumor tissues.Furthermore,the results also showed that 3-MA reversed the proliferation,migration and invasion of RCC cells inhibited by HOTTIP silencing,and RAPA also reversed the proliferation,migration and invasion of RCC cells promoted by HOTTIP over-expression.Conclusions Lnc RNA-HOTTIP can affect the progression of RCC by regulating autophagy.Part 3 Lnc RNA-HOTTIP regulates the molecular mechanism of RCC autophagyObjective To further explore the specific molecular mechanism involved in the regulation of HOTTIP on RCC autophagy.Methods In this research,we firstly measured the formation of autophagosomes and the expression of autophagy-related genes in the stably transfected RCC cell lines constructed above.On the basis,we further examined the expression of proteins involved in the autophagy-related pathway.Results The results showed that HOTTIP silencing significantly induced the formation of autophagosomes,while HOTTIP over-expression significantly inhibited the formation of autophagosomes.Moreover,the results also showed that HOTTIP silencing significantly increased the gene and protein expression level of Atg13,and significantly reduced the phosphorylated levels of PI3 K and Akt,while HOTTIP over-expression significantly reduced the gene and protein expression level of Atg13,and significantly increased the phosphorylated levels of PI3 K and Akt.Conclusions Lnc RNA-HOTTIP can regulate RCC autophagy through the PI3K/Akt/Atg13 signaling pathway.