RNA Interferencing Krüppel Like Factor 8 Inhibits The Renal Carcinoma 786-0 Cells Growth In Vitro And In Vivo

Renal cell carcinoma (RCC) is the common genitourinary tract tumor andaccounts for approximately 2% of adult cancer.Radical nephrectomy is the mosteffective treatment for localized renal cell carcinoma.RCC remains the highmortality due to the lack of early detection methods and effective treatmentsfor late-stage cancers.Twenty-five percent of patients were diagnosed withlocally invasive or metastatic RCC.The lack of sensitive efficacy ofchemotherapy and radiation therapy in late-stage RCC has led to a 5-year survivalranging from 5% to i0%.The use of recombinant human interleukin-2 (IL-2) andrecombinant human interferonα(IFN-α), either alone or in combination,however, is limited by their toxicity and generally poor overall responserates.Understanding the molecular mechanisms underlying RCC progression andmetastasis is important for developing new strategies for early diagnosis andtherapies required for improvement of patient survival.Kruppel like factor 8 ( KLF8 ) was one of Kruppel like transcription factorfamily.Over-expressed KLF8 has recently been found in some of human malignanttumors,while it is low expressed in the normal tissues.KLF8 has been thoughtas a key transcription factor downstream of FAK in the regulation of the cellcycle by activating cyclin D1 gene promoter and also plays a critical role inoncogenic transformation,cell invasion and epithelial to mesenchymaltransition (EMT). Especially,whether and how KLF8 might play a role in RCCtumorigenesis,progression and invasion is little known.In the present study,we firstly investigated the expression of KLF8 mRNAand protein in 42 sporadic RCC samples by reverse transcription polymerase chainreaction (RT-PCR) and immunohistochemistry(IHC),explored the correlation ofKLF8 and the carcinogenesis with development of human renal cell carcinoma.Subsequently we constructed small interference RNA (siRNA) sequences targetingKLF8, transfected it into the human RCC cell line 786-0, identified thatKLF8-siRNA can inhibited expression of KLF8 mRNA and protein leveleffectively.Further,we explored changes in cell growth, invasiveness,cellcycle and cell apoptosis,studied the influence on the cyclin Dl and FAK,E-cadherin mRNA and protein when KLF8-siRNA transfected 786-0 cells.Inoculating the renal carcinoma cells and interference renal carcinomacells to the nude mice,the influence of KLF8 genes on the oncogenicity wasinvestigated.In this project,using the KLF8-siRNAmethod to study the influenceon the proliferation of renal carcinoma 786-0 cells and the development of tumor,providing the experimental evidences to the gene therapy of renal cellcarcinoma.PartⅠThe expression KLF8 in renal cell carcinomaand clinical significancesObjectives: To investigate the expression of KLF8inrenal cell carcinomaand clinical significances.Methods:We firstly investigated the expression of KLF8 mRNA and proteinin 42 sporadic RCC samples and adjacent non-tumorous renal tissues by RT-PCRand IHC.Results:RT-PCR results revealed that the relative expression of KLF8 mRNAin RCC and adjacent non-tumorous renal tissues was (0.869±0.321) and (0.416±0.117) respectively and statistically significant difference exists betweenthem (P＜0.001).KLF8 protein expression was positive in 36 cases of RCC tissues(36/42,85.7%),higher than that in adjacent non-tumorous tissues (16/42,38.1%,P＜0.001).The high levels of KLF8 mRNA and protein expression were relatedwith the larger tumor size (P＜0.001) and high clinical stage(P＜0.001),but notrelated with sex,age and cell differentiate (P＞0.05).Conclusions:The KLF8 mRNA and protein expression were elevated in thehuman RCC.KLF8 was involved in the development and progression of RCC,whichmay be used as a target of renal cell carcinoma gene therapy.PartⅡThe construction and identification of RNA interferencingKLF8 sequences Objectives:To investigate the construction and identification of RNAinterferencing KLF8 sequences.Methods:Designing and synthesizing the siRNA which can inhibit the KLF8expression and transfecting with renal cancer cell 786-0. Checking theexpression level of KLF8 mRNA and protein by RT-PCR and Western blot.We wantedto select a most efficient siRNA sequences for further study.Results:Compared with blank control (0.977±0.003) and scrambled siRNA(0.917±0.014),KLF8-siRNA significantly inhibited the relative expression ofKLF8 mRNA(P＜0.001): siRNA1 (0.276±0.017), siRNA2 (0.084±0.005), siRNA3(0.316±0.014). SiRNA2 was identified as the most potent sequence whichsuppressed KLF8 mRNA expression.Western blot analysis also demonstrated a significant reduction in KLF8protein level(P＜0.001):siRNA1 (0.491±0.012),siRNA2 (0.264±0.029), siRNA3(0.401±0.013).when compared with that in blank control (0.822±0.016) and thatin scrambled siRNA (0.807±0.019).There was no statistically significancesbetween the expression of KLF8 mRNA and protein in blank control group and thatin scrambled siRNA group (P＞0.05).In agreement with RT-PCR results, the expression of KLF8 protein wasdown-regulated effectively by siRNA2.Conclusions: Constructing KLF8-siRNA can effectively inhibit theexpression KLF8 mRNA and protein in renal cancer cell 786-0.Subsequentexperiments focused on the siRNA2 because it was the most effective at inhibitingKLF8 mRNA and protein expression.PartⅢRNA interferencing KLF8 inhibits the renal carcinoma786-0 cells growthin vitroObjectives:To investigate RNA interferencing KLF8 inhibits the renalcarcinoma 786-0 cells growth in vitro.Methods:KLF8-siRNA2 were transfected with renal carcinoma cell 786-0.Checking the expression level of cyclinD1 and FAK,E-cadherin mRNA and protein by RT-PCR and Western blot.The effects of siRNA targeting KLF8 on growth,invasiveness,cell cycle and cell apoptosis of 786-0 ceils were evaluated byMTT Assay,Matrigel Invasion Assay and flow cytometry in vitro.Results: Compared with blank control (0.693±0.009),(0.487±0.020)and scrambled siRNA group(0.715±0.018),(0.490±0.012), the relative mRNAexpression of cyclin D1 (0.217±0.018) and FAK (0.113±0.012) wassignificantly in KLF8-siRNA2 group(P＜0.001).The relative protein expression of cyclin D1 (0.217±0.018) and FAK(0.113±0.012) was significantly in KLF8-siRNA2 group (P＜0.001),when comparedwith blank control (0.780±0.017),(0.810±0.021) and scrambled siRNAgroup(0.607±0.024),(0.593±0.008).There was no statisticallysignificancesbetween that the expression ofcyclin D1,FAK mRNA and protein in blank controlgroup and that in scrambled siRNA group (P＞0.05).The relative mRNA and protein expression of E-cadherin was alsosignificantly up-regulated in KLF8-siRNA2 group.MTT assay showed that thegrowth of 786-0 cell in KLF8-siRNA2 group are slower than that of blank controland scrambled siRNA group (P＜0.05).Migrating numbers of cells was significantlyinhibited in KLF8-siRNA2 group by Matrigel Invasion Assay.KLF8-siRNA2 groupinduced an increase in G0/G1 phase cells(P＜0.001),decreased in Sphase(P＜0.001),the percentage of apoptotic cells in KLF8-siRNA2 group wereincreased(P＜0.001).Conclusions:The siRNAi targeting KLF8 can significantly decrease theexpression mRNA and protein of cyclinD1 and FAK.Down-regulation KLF8 expressioncan induce up-regulation the expression mRNA and protein of E-cadherin.It can decrease cell grow,suppressing cell invasion,affecting the cellcycle,increasing the percentage of cell apoptosis.KLF8 may play a important rolein promoting cell grow, maintainting cell invasion,mediating cell cycle,suppressing cell apoptosis.KLF8 may be involved in the carcinogenesis andprogression.It may be a key factor of FAK mediating in the regulation of cellcycle and dual regulation may be existed between KLF8 and FAK.Blocking KLF8expression by RNAi might inverse EMT in the renal cancer cells. RNA interferencing KLF8 inhibits the renal carcinoma 786-0 cellsgrowth in vivoObjective:Exploring the influence of RNA interferencing KLF8 inhibitthe renal carcinoma 786-0 cells oncogenicity in vivo.Methods:18 Male Balb/c male nude mice were randomly divided into blankcontrol group,scrambled siRNA group,KLF8-siRNA2 group(n=6).Tumor wasimplanted by subcutaneous injection of 3.5×10~6 cells/mouse in 200μl of a50/50 dilution of RPMI 1640 in PBS into the left flanks of the nude mice.Whentumor nodules were growed ,equal volume of PBS was used for intratumorinjection as blank control group.Scrambled siRNA group received intratumorinjections (50μg/ mouse ) of scrambled siRNA and KLF8-siRNA2 group receivedintratumor injection of 50μg KLF8-siRNA2 every 3 day.All mices were executedin 6 weeks after the inoculation,stripped and put out the tumor which to beweighed and then calculated the tumor volume according to the formula.RI-PCRand western blot were used to determine the expression of KLF8 mRNA and proteinin three group.Results:Tumor weight in KLF8-siRNA2 group (2.073±0.067)g was alsosignificantly decreased (P＜0.001) than that in blank controlgroup(3.317±0.144)g and in scrambled siRNA group(3.333±0.185)g after sixweeks,while there was no statistically significance between blank controlgroup and scrambled siRNA group (P＞0.05).Compared with blank control (0.488±0.021) and scrambled siRNA group(0.483±0.017), the relative mRNA expression of KLF8 (0.269±0.028)wassignificantly in KLF8-siRNA2 group(P＜0.001).The relative protein expressionof KLF8(0.278±0.044) was significantly in KLF8-siRNA2 group(P＜0.001),whencompared with blank control (0.785±0.016)and scrambled siRNAgroup(0.731±0.023).There was no statistically significances between that theexpression of KLF8 mRNA and protein in blank control group and that in scrambledsiRNA group (P＞0.05).Conclusions:After transfectd with KLF8-siRNA2,the formation anddevelopment of renal tumor model can be obviously inhibited.The expressionof KLF8 mRNA and protein in KLF8-siRNA2 group was also decreased.It suggestedthat KLF8 may play an important role in the carcinogenesis and progression.