Effects Of Long Non-coding RNA-linc00645 On Proliferation And Migration Of Clear Cell Renal Carcinoma Cells

BACKGROUND AND PURPOSERenal cell carcinoma,one of the most common cancers of the urinary system,kills about 100,000 people worldwide each year,accounting for 5 percent and 3 percent of malignancies in men and women,respectively.Renal cell carcinoma is a malignant tumor originated from renal parenchymal urinary tubular epithelial cells,which can be divided into several cell subtypes.Clear cell renal carcinoma is the most common pathological type of renal carcinoma.There is no simple,inexpensive screening method for renal cell carcinoma.At present,the preoperative diagnosis of renal cancer mainly relies on imaging examination,but it is sometimes difficult to determine the benign and malignant renal tumors by imaging examination.Diagnosis mainly depends on postoperative pathological examination.At present,radical nephrectomy and partial nephrectomy are the main treatments for renal carcinoma.There is no effective adjuvant treatment after the operation.Although cytokine therapy has been used in clinical practice for several years,it is sometimes difficult to obtain satisfactory results due to its large side effects and low effective rate.About 40%of patients develop tumor recurrence and metastasis after surgery,and the prognosis of patients with metastatic renal cancer is very poor,with a 5-year survival rate of about 10%.Although there are currently targeted therapies for metastatic renal carcinoma that can improve overall survival by several months.But the cost is high,long-term application still has the malpractice of poor tolerance,drug resistance.Therefore,it is urgent to study new molecular biological targets to guide early diagnosis and follow-up treatment.Long non-coding RNA(lncRNA)is a type of RNA that is more than 200 nucleotides long and cannot encode proteins.In recent years,with the popularization of second-generation sequencing technology and the mature application of relevant research technologies,lncRNA has been found to play an important role in a variety of physiological and pathological processes and play a crucial role in the occurrence and development of a variety of diseases.Long non-coding RNA-linc00645 is an intergenic IncRNA located on human chromosome 14q12.Its expression has tissue specificity,and the expression amount in normal renal tissues is much higher than other tissues and organs.Previous literature has reported that its expression in renal carcinoma is significantly down-regulated compared with normal renal parenchyma.However,its specific mechanism of action in renal carcinoma is still unclear.We intend to elaborate its specific effect on clear renal cell carcinoma cells and its mechanism of action through cell function experiment and downstream mechanism study.METHOD1.Analyze the expression of linc00645 in TCGA and GTEx data.2.Clinical information of renal cancer patients admitted to the urology department of our hospital from 2015 to 2017,as well as samples of renal cancer tumors and para-cancer normal tissues were collected,and PCR experiment was conducted to verify the expression of linc00645 in tumor tissues and para-cancer normal tissues.3.The subcellular localization of linc00645 was verified by 786-O and ACHN cell lines.4.CCK-8 assay was performed to verify the effect of linc00645 overexpression on the proliferation of clear renal cell carcinoma cells.5.Annexin V-PI double staining flow cytometry was used to investigate the effect of linc00645 overexpression on apoptosis of clear renal cell cells.6.Transwell assay was used to verify the effect of linc00645 overexpression on the migration of clear renal cell carcinoma cells.7.Investigate the RNA binding protein of linc00645 in CHIRP-MS experiment.8.PCR and WB experiment were conducted to explore the influence of linc00645 overexpression on the mRNA and protein expression of downstream PKM2.RESULT1.The expression of linc00645 is tissue specific and is much higher in the kidney than in other normal tissues and organs.Its expression was significantly lower in clear cell carcinoma of the kidney than in para-tumor normal tissue.2.Linc00645 is distributed in the nuclei and cytoplasm of renal clear cell carcinoma cells.3.The overexpression of linc00645 can significantly inhibit the proliferation and migration of 786-O and ACHN cells,and promote the apoptosis of renal cancer cells.4.Linc00645 can form specific binding with 48 proteins such as ENO1,LDHA and PKM2 etc.5.The overexpression of linc00645 can inhibit the expression of PKM2 protein.CONCLUSIONThe expression of linc00645 in clear cell renal carcinoma was lower than that in para-carcinoma normal tissue,and it was distributed in both nucleus and cytoplasm.Linc00645 can reduce the expression of PKM2 in clear cell renal carcinoma cells,inhibit the proliferation and migration of tumor cells,and promote cell apoptosis,so as to play the role of tumor inhibition.Linc00645 can provide reference for the diagnosis of renal clear cell carcinoma,and Linc00645 and PKM2 are expected to be therapeutic targets for clear cell renal carcinoma..