TET1 inhibits the migration and invasion of cervical cancer cells by regulating autophagy

Figures & data

Table 1. The primer sequence used in this study.

Gene name	Primer type	Primer Sequence
GAPDH	Forward	GGAGCGAGATCCCTCCAAAAT
	Reverse	GGCTGTTGTCATACTTCTCATGG
E-cadherin	Forward	TACACTGCCCAGGAGCCAGA
	Reverse	TGGCACCAGTGTCCGGATTA
Vimentin	Forward	GACGCCATCAACACCGAGTT
	Reverse	CTTTGTCGTTGGTTAGCTGGT
Snail	Forward	GAGGCGGTGGCAGACTAG
	Reverse	GACACATCGGTCAGACCAG
NKRF	Forward	AGCCAGCTCAAATTGTAGTCT
	Reverse	ACCTTATCTTGTTTGTCGCCA
HIST1H2AK	Forward	TTTTGAGACAAGGCCTGGC
	Reverse	CCTGGTAGGCGGAGTTTACA
PGRMC2	Forward	GCAGGATTAGCTGCTTCTTTTA
	Reverse	TCAGGGAGATATGATTAGAGATAGAAT
AHCYL2	Forward	CCTGACCTTGTGATCCACCT
	Reverse	TCACACCTGTAATCCCAGCA
IFT27	Forward	GAGCACTGATCACCATCGAA
	Reverse	GCAAGGCACACATCACCTAA
PARD3B	Forward	ATGGGTTGCACTAGCAGAGG
	Reverse	CCCAGCTCTTCTTGCTCTGA
CHSY1	Forward	CCTCGTTACTTCTGGGTTGC
	Reverse	CCATGCTTTCTGTGTGCAGT
GRAMD3	Forward	GCACTGCATTTGTTTGTTTTCA
	Reverse	TGGATTTACTGCAGTGCCTTT
INSIG2	Forward	GGAGCTGAAATTGTGGCAATA
	Reverse	GAATTCGGCTGTGAATCCAT
PALB2	Forward	TTTCCCATCCATGACTCAGA
	Reverse	TCATGACCGTTGTTTGAACC
ZNF558	Forward	GGCAGAGCTGGTCTCTTCAG
	Reverse	ACTGACTCCTGGGTTGATGG
AHCYL2	Forward	TACCGATACACAGCCATCCA
	Reverse	AGAGTGGGAAAGGCAAAGTG
ATXN10	Forward	CTTACAATGGCCCTGCATTT
	Reverse	CATCTGCATTTTCAGCAGGA
PRKAG2	Forward	CTTGGAGATGCAGGAGAAGG
	Reverse	AGGTCTGGAGAAGCGGAGTT
si-NKRF1	sense	GGCUUGGUCUGGAUGUAGA
	antisense	UCUACAUCCAGACCAAGCC

Figure 1. TET1 expression was downregulated in the cervical cancer tissues. (a) The expression of TET1 in the cervical cancer tissues with different degrees of differentiation; (b) The TET1 expression in cervical cancer and normal cervical tissues; (c) TCGA data indicating that TET1 was downregulated in cervical cancer.

Figure 2. TET1 knockdown promoted the migration and invasion of cervical cancer cells.

(a,b) Verification of TET1 knockdown and overexpression in cervical cancer SiHa and HeLa cells; (c,d) The effect of TET1 knockdown on the migration of cervical cancer SiHa and HeLa cells was detected by scratch assay; (e) Transwell assay was performed to detect the effect of TET1 knockdown on the invasion ability of cervical cancer SiHa and HeLa cells; (f) The effect of TET1 overexpression on the migration of cervical cancer SiHa and HeLa cells was detected by scratch assay; (g) Transwell assay was performed to detect the effect of TET1 overexpression on the invasion ability of cervical cancer SiHa and HeLa cells.

Figure 3. TET1 reduces the proliferation, clonal formation, and tumorigenesis of cervical cancer cells.

(a,b) The knockdown of TET1 promoted the colony formation of SiHa and HeLa cells; The overexpression of TET1 impaired the colony formation of SiHa and HeLa cells; (c,e) The effects of TET1 knockdown or overexpression on the proliferation of cervical cancer cells were detected by cell cycle(C), RTCA (d)and subcutaneous tumorigenesis(E)

Figure 4. Effect of TET1 on epithelial-mesenchymal transformation (EMT) of cervical cancer SiHa cells.

(a,b) Western blotting was performed to detect the expression changes of EMT-related indicators after the knockdown or overexpression of TET1 in SiHa cells; (c,d) RT-PCR was performed to detect the expression changes of EMT-related indicators after the knockdown or overexpression of TET1 in SiHa cells.

Figure 5. Effect of TET1 on the biological functions of cervical cancer cells.

(a) KEGG analysis revealed the signalling pathways associated with the upregulated activity of TET1 knockdown in SiHa cells when compared with that in wild-type cells; (b) KEGG analysis was performed to identify the signalling pathways associated with the downregulated activity of TET1 in SiHa cells when compared with that in wild-type cells. (c) Western blotting was performed to detect the autophagy indexes of SiHa cells after TET1 knockdown; (d,e) Immunofluorescence also showed that there were more autophagosomes in the TET1 cas9 group than in the WT group, while the TET1^+/+ group had the least number of autophagosomes. Furthermore, immunofluorescence results show that knockdown of TET1 can attenuate the inhibitory effect of 3 MA on autophagy, while overexpression of TET1 can continue to inhibit autophagy in the presence of 3 MA; (f) Immunohistochemical detection of LC3 and P62 expression in cervical cancer and adjacent tissues.

Figure 6. Effects of autophagy on TET1-mediated biological changes in cervical cancer cells.

(a – c) 3 MA to treat SiHa cells, and the results suggested that the proliferation, invasion, and migration of SiHa cells in the 3 MA group were significantly lower than those in the control group; (d,e) TET1 knockdown could inhibit this effect of a 3 MA-induced decrease in the invasion and migration abilities.

Figure 7. Effect of TET1 on the gene methylation level and mRNA expression.

(a,b) Immunohistochemistry was performed to detect the expression of 5 mC and 5 hmC in highly and poorly differentiated Normal cervical tissue cervical cancer tissues; (c,d) Genes with a high methylation level, but low mRNA expression and genes with a low methylation level but high mRNA expression were observed through methylation sequencing. Among them, only NKRF and HIST1H2AK were involved in the change of the promoter methylation level; (e,f) The specific methylation levels of the selected genes were detected by MEDIP-qPCR, and the NKRF and HIST1H2AK with upregulated promoter methylation and downregulated mRNA verified by q-PCR; (g) The correlation among TET1, NKRF, and HIST1H2AK was analysed with reference to the TCGA database. (h) The knockdown efficiency of NKRF was verified by RT-qPCR; (i) The expression of autophagy related protein LC3B was detected after NKRF knockdown.