Intervention Study Of KiSS-1 Gene Knockout On The Metastatic Behavior Of Human Low Metastatic Nasopharyngeal Carcinoma Cell Line

Objective: To study the effect of KiSS-1 gene on metastatic ability of nasopharyngeal carcinoma(NPC)with low metastatic cell line SUNE-1-6-10 B after being knocked out.Methods:(1)KiSS-1 gene was knocked out in the NPC cell line SUNE-1-6-10 B and the NPC cell line with low expression of KiSS-1 gene was obtained;(2)The cells of NPC were injected into nude mice respectively before and after KiSS-1 gene knockedout.Observe the effect of KiSS-1 gene on both armpit tumor and its metastasis.Results: KiSS-1 gene in NPC cell line SUNE-1-6-10 B was successfully knocked out by CRISPR/Cas9 technique.The NPC cell line with low expressing KiSS-1 gene was obtained.The DNA of KiSS-1 gene was extracted from NPC cell line to conduct SURVEYOR verification.The PCR products of DNA fragment was digested by T7 E I enzyme,generating two other fragments(231bp and 190 bp fragments).It was showed that the PCR product can not be fully matched during the slow annealing,so that it can be identified by the enzyme and be cut into two small DNA pieces.It was confirmed that the succeed in causing fixed point mutation in the SUNE-1-6-10 B cell genome by CRISPR/Cas9 gene editing.Kisspeptin protein(KiSS-1 gene expression product)was extracted to carry out Western blot verification.The results showed that the Kisspeptin protein was significantly lower expressed in the NPC cell with KiSS-1 gene knockout.As an experimental group,the NPC cells with KiSS-1 gene knockout were injected into armpit of nude mice.Each nude mouse was injected with 2 ml concentration of 5×106/mL cell suspension.The same concentration and volume of NPC cells(SUNE-1-6-10B)was injected into mice armpit,which was used as a control group.The results showed that experimental group and the control group tumor weight were 6.72 ± 0.37 g and 6.80 ± 0.38 g respectively.There was no significant difference between two sets of data(P>0.05).The lung metastases formation rate in the Experimental group and control group was 40% and 10% respectively,with significant difference between the two sets of data(P<0.05).Conclusion: CRISPR / Cas9 technology can be used to knockout KiSS-1 gene in NPC cell line SUNE-1-6-10 B which has low metastatic habits.KiSS-1 gene can inhibit metastasis of NPC cells.