The Influence Of RhoC On Human Hep2Laryngeal Squamous Carcinoma Cell

Objectives：By delivering exogenous gene into Hep2laryngeal squamous carcinomacell line, we alternatively repressed and strengthened the expression of RhoC. Afterwhich，we observed what would happen to the morphology, the apoptosis and theexpression of cancer stem cell(CSC) marker ALDH1A1in these cells, at last, make aconclusion of the influences of RhoC on biological behavior and CSCs in the laryngealsquamous carcinoma, which will provide experimental evidences for therapeuticallytargeting RhoC in controlling metastasis of laryngeal squamous carcinoma(LSC).Methods: Design and synthesize two kinds of recombinant plasmids, one can formRhoC-shRNA, which own the ability to influence the expression of RhoC, another onecan form constitutively active RhoC, as a result, strengthen the expression of RhoC. Werespectively transfecte Hep2cells with these two recombinant plasmids by liopsom, atthe same time, optimize transfection conditions, through which we achieve the mostproper transfected efficiency. Whereafter, We measure the mRNA of CSC markerALDH1A1with QPCR, tested the apoptosis with TUNEL reactivity, visualize tumorcells shape by staining cell skeleton with Alexa fluor phalloidin, test motility byscratching method.Results:(1)Through recombinant plasmid sequencing and transfecting recombinantplasmid into293T cells by liopsom, in which recombinant plasmid express EGFP, wecertified that the GW-shRNA recombinant plasmid, which can repress the expression ofRhoC, and the13GS03recombinant plasmid, strengthen RhoC expression, had beensuccessively synthesized.(2)Under the culture condition with24-well plates, using1.2μｇ recombinant plasmid GW-shRNA or13GS03, and mixing with4.2μl liopsom, wecould get the most proper transfected efficiency.(3)After repressing the expression of RhoC in Hep2cell line, the expression of CSC marker ALDH1A1was significantlydecreased. The apoptosis of cancer cells was elevated. The cells became larger, hadpolygonal shapes.(4)After improving the expression of RhoC in Hep2cell line, theexpression of CSC marker ALDH1A1and apoptosis had no significant differences withblank control cells, While led to spindle-shaped morphology, resembling mesenchymalcells， which implied an increased capability of motility.(5)Wound healing assayrevealed that inhibiting the the expression of RhoC, we could slow down Hep2cellsmotility, while RhoC over-expression can drive cells motility.Conclusion:1.GW-shRNA recombinant plasmid which interfere with the expression ofRhoC and13GS03recombinant plasmid which strengthen RhoC are successfullyconstructed.2.RhoC play a role in initiation of metastasis by regulates CSC in LSC.3.RhoC have anti-apoptosis effect on LSC, Which make cancer cells survival in thecirculation system and arrest in a distant target organ.4.RhoC have a positive role inLSCC metastasis.Above all, RhoC is a promising target of anti-metastases in LSC.