Downregulation Of Rack1Expression Inhibits Proliferation,Migration And The Invasion Capacities Of Mouse Hepatocellular Carcinoma Cells

Background: Death due to malignant tumors sill accounts for majority ofhuman causes of death. It is the first or second in worldwide mortality. Eachyear about7000000people die of cancer, and the incidence is clearly on theupward trend. Primary liver cancer is the most common digestive systemmalignancy. Morbidity and mortality of primary liver cancer is in the topfive, and is a serious threat to human health.The incidence of hepatocarcinoma ranked sixth in the world at present, about600000people get hepatocarcinoma in the world each year. Hepatocarcinomaranks third in mortality rates. High invasion rate, early metastasis, relapse rateafter treatment, and poor prognosis are the clinical and pathologicalfeatures of Liver cancer.Liver cancer is a malignant epithelial tumor. Lymph node metastasis isone of the main ways of early metastasis, and also one of the key factorsinfluencing the prognosis. However, the mechanism remains unclear, and itis still difficult to treat. Early detection and prevention of HCC metastasishas become the key to improving the survival rate of patients.Hca-F and Hca-P is a pair of syngenetic mouse hepatocarcinomaascites cell lines which have different potential of lymphatic metastasis.Hca-F with a high lymphatic metastasis rate of70%, and Hca-P with a lowlymphatic metastasis rate of30%have been proved to be an ideal cell modelfor studying the lymphatic metastasis.The expression of Receptor of activated C-kinase1（receptor of activated C-kinase1, Rack1）, also known as Gnb2L1, differed significantly at the of mRNA andprotein levels. Rack1is a scaffolding protein inside the cell, which was firstdiscovered in1991and received a major concern due to the possibility of participating in a variety of biological functions involving tumor regulation.However, there is no clear comprehensive research report about the correlationbetween the gene and the biological behaviour of hepatocellular carcinoma （HCC）, orthe role the gene plays in migration, invasion, metastasis or molecular mechanisms ofliver cancer.Objective: Short hairpin RNA （shRNA）-Rack1expression plasmidswere constructed and stably transfected into the cells to study the effect tocell proliferation, migration, and invasion of the Rack1down-regulatedmouse hepatocarcinoma cells.Method:1. Four shRNAs that targeted different locus of the mouseRack1gene （NM₀08143.3） were designed together with a control shRNA.Four PGPU6/GFP/Neo-shRNA-Rack1expression plasmids wereconstructed， and all the expression plasmids were confirmed by DNAsequence analysis.2. The levels of expression of Rack1mRNA and protein were detectedby quantitative real time polymerase chain reaction （qRT-PCR） and WBanalysis, respectively. The shRNA-Rack1-292-Hca-F cells were chosen forfurther experiments.3. Cells were divided into three groups:（1） shRNA-Rack1-292-Hca-F;（2） control shRNA-Hca-F;（3） unmanipulated Hca-F cells were used ascontrol. Cell proliferation was assessed by Cell counting kit-8（CCK8）assay according to the protocol of the manufacturer. Using the CCK8,absorbance values were determined at450nm wavelength, which indirectly reflectsthe number of living cells and the proliferation ability was detected by analyzing thedifferences in the absorbance values.4. Cell migration assay and invasion assay were measured using theBoyden-transwell assay （8-μm pore size）. The upper chamber was serum-free culturewhilst the lower chamber contained serum to serve as a chemoattractant and the cellsthat passed through the filter were stained and counted to know which cell group hasthe highest migration and invasion ability.Results:1. Endonuclease activity assays and DNA sequence analysis confirmedthe recombinant plasmids of shRNA-Rack1.2. The levels of mRNA expression of Rack1inshRNA-Rack1-292-Hca-F and shRNA-Rack1-1105-Hca-F were decreased by70%and52%, respectively, quantified by qRT-PCRanalysis compared to Hca-F cells. WB results indicatedshRNA-Rack1-292-Hca-F and shRNA-Rack1-1105-Hca-F reduced theEch1protein levels by44%and34%compared to Hca-F cells, butnonspecific-sequence control shRNA transfected Hca-F cells showedno difference compared to Hca-F cells. The shRNA-Rack1-292-Hca-Fwas more efficient than the others, so this was chosen as theshRNA-Rack1-Hca-F for further experiments.3. Cell proliferation: Cell counting kit-8（CCK8） test results showedthat down-regulating the expression of Rack1results in significantdecrease in cell proliferation capacity.4. Transwell cell migration and invasion: experimental results showedthat downregulation of Rack1reduced cell migration and invasion.Conclusion:1. PGPU6/GFP/Neo-shRNA-Rack1expression vector was constructedand transfected into Hca-F cells. Gene expression of Rack1stabledown-regulated Hca-F cell lines proved that Rack1wasdownregulated successfully.2. The shRNA-Rack1-292-Hca-F was more efficient than the othersthrough the WB and qRT-PCR analysis, so this was chosen as theshRNA-Rack1-Hca-F for further experiments.3. The reduction in Rack1expression showed a decrease in Hca-F cellproliferation, migration, and invasion.