The downregulation of microRNA-375 in human cervical squamous cell carcinoma promotes invasion and migration by targeting JAK2

Micro RNAs (miRNAs) contribute to the progression and expansion of many cancers, including cervical cancer. Reports reveal the down-regulation of MicroRNA-375 (miR-375) within cervical cancer tissues. Nevertheless, the mechanism for regulation of cervical cancer progression by miR-375 is still unknown. The current work deals with the molecular mechanisms orienting the regulatory impacts of miR-375 on invasion and migration in human cervical carcinoma. miRNA expression within cervical cancer tissues was assessed through Stem-loop RT-PCR. The invasion and migration of miR-375 mimic-transfected SiHa cells were observed by the Transwell test and wound healing test, respectively. Bioinformatics analysis was performed for predicting the target gene of miR-375, which was validated by the dual-luciferase test. Real-time RT-PCR and Western blot analysis were utilized for detecting protein expression and mRNA transcription. In cervical cancer, miR-375 expression was 4.2 ± 0.15-fold lower compared to the adjacent noncancerous tissues. The number of invasive and migrated SiHa cells (32 ± 3.6; 21.86 ± 6.4) transfected with miR-375 mimics were considerably lower in comparison with the NC group (101 ± 7.5; 41 ± 5.6) after 24 hours. Real-time RT-PCR and Western blot analysis confirmed JAK2 as a downstream target gene of miR-375. The protein expression and mRNA level of JAK2 in miR-375 mimic-transfected SiHa cells were decreased. The present study indicated that the invasion and migration of SiHa cells are regulated by miR-375 through down regulating JAK2 expression. This study provides a theoretical and experimental basis for reversing the metastasis of cervical cancer and developing new candidate drugs.

miR-375; Cervical squamous cell cancer; Invasion; Migration; JAK2.

[1] Wang F., Li Y., Zhou J.S., Xu J.F., Peng C.J., Ye F., et al.: “miR-375 is down-regulated in squamous cervical cancer and inhibits cell migration and invasion via targeting transcription factor SP1”. Am. J. Pathol., 2011, 179, 2580-2588.

[2] Jemal A., Bray F., Center M.M., Ferlay J., Ward E., Forman D.: “Global cancer statistics”. CA Cancer J. Clin., 2011, 61, 69-90.

[3] Ding H., Wu Y.L., Wang Y.X., Zhu F.F.: “Characterization of the microRNA expression profile of cervical squamous cell carcinoma metastases”. Asian Pac. J. Cancer Prev., 2014, 15, 1675-1679.

[4] Lechanteur A., Furst T., Evrard B., Delvenne P., Piel G., Hubert P.J.M.P.: “Promoting vaginal distribution of E7 and MCL-1 siRNA-silencing nanoparticles for cervical cancer treatment”. Mol. Pharm., 2017, 14, 1706-1717.

[5] Makino H., Nishio S., Tsubamoto H., Shimada M., Nishikawa R., Kai K., et al.: “Treatment and prognosis of bone metastasis from cervical cancer (KCOG-G1202s)”. J. Obstet. Gynaecol. Res., 2016, 42, 701- 706.

[6] Onal C., Erbay G., Guler O.C.: “Treatment response evaluation us-ing the mean apparent diffusion coefficient in cervical cancer patients treated with definitive chemoradiotherapy”. J. Magn. Reson. Imaging, 2016, 44, 1010-1019.

[7] Yamagami W., Aoki D., Research G.: “Annual report of the Committee on Gynecologic Oncology”. Japan Soc. Obstet. Gynecol., 2016, 41, 1861.

[8] Ning M.S., Ahobila V., Jhingran A., Stecklein S.R., Frumovitz M., Schmeler K.M., et al.: “Outcomes and patterns of relapse after definitive radiation therapy for oligometastatic cervical cancer”. Gynecol. Oncol., 2018, 148, 132-138.

[9] Barukčić I.: “Human Papillomavirus-the cause of human cervical cancer”. J. Biosci. Med., 2018, 6, 106.

[10] Shader R.I.: “Cervical cancer and human papilloma virus”. Clin Ther., 2016, 38, 429.

[11] Berman T.A., Schiller J.T.: “Human papillomavirus in cervical cancer and oropharyngeal cancer: One cause, two diseases”. Cancer, 2017, 123, 2219-2229.

[12] Liu B., Ding J.F., Luo J., Lu L., Yang F., Tan X.D.: “Seven protective miRNA signatures for prognosis of cervical cancer”. Oncotarget, 2016, 7, 56690-55698.

[13] Li S., Yang F., Wang M., Cao W., Yang Z.: “miR-378 functions as an onco-miRNA by targeting the ST7L/Wnt/β-catenin pathway in cervical cancer”. Int. J. Mol. Med., 2017, 40, 1047-1056.

[14] Gao D., Zhang Y., Zhu M., Liu S., Wang X.: “miRNA Expression Profiles of HPV-Infected Patients with Cervical Cancer in the Uyghur Population in China”. PLoS One, 2016, 11, e0164701.

[15] Xia Y.F., Pei G.H., Wang N., Che Y.C., Yu F.S., Yin F.F., et al.: “miR-3156-3p is downregulated in HPV-positive cervical cancer and performs as a tumor-suppressive miRNA”. Virol. J., 2017, 14, 20.

[16] Li C.M., Zheng J.G. and Du G.S.: “miRNA: a new regulator of gene expression”. Hereditas (Beijing), 2004, 26, 133-136. (In Chinese)

[17] Lu J., Getz G., Miska E.A., Alvarez-Saavedra E., Lamb J., Peck D., et al.: “MicroRNA expression profiles classify human cancers”. Nature, 2005, 435, 834-838.

[18] Sheng X.H., Du L.X.: “Progress on the research of microRNAs and its function in humans and animals”. Hereditas, 2007, 29, 651-658.(In Chinese)

[19] Zhang X., Yan Z., Zhang J., Gong L., Li W., Cui J., et al.: “Combination of hsa-miR-375 and hsa-miR-142-5p as a predictor for recurrence risk in gastric cancer patients following surgical resection”. Ann. Oncol., 2011, 22, 2257-2266.

[20] Huang X., Yuan T., Liang M., Du M., Xia S., Dittmar R., et al.: “Exosomal miR-1290 and miR-375 as Prognostic Markers in-Castration-resistant Prostate Cancer”. Eur. Urol., 2015, 67, 33-41.

[21] Yan J.W., Lin J.S., He X.X.: “The emerging role of miR-375 in cancer”. Int. J. Cancer, 2014, 135, 1011-1018.

[22] Selth L.A., Das R., Townley S.L., Coutinho I., Hanson A.R., Centenera M.M., et al.: “A ZEB1-miR-375-YAP1 pathway regulates epithelial plasticity in prostate cancer”. Oncogene, 2016, 36, 24-34.

[23] Yu X., Zhao W., Yang X., Wang Z., Hao M.: “miR-375 affects the proliferation, invasion, and apoptosis of HPV16-positive human cervical cancer cells by targeting IGF-1R”. Int. J. Gynecol. Cancer, 2016, 26, 851-858.

[24] Liu S., Song L., Yao H., Zhang L., Xu D., Gao F., et al.: “MiR- 375 is epigenetically downregulated by HPV-16 E6 mediated DNMT1 upregulation and modulates EMT of cervical cancer cells by suppressing lncRNA MALAT1”. PLoS One, 2016, 11, e0163460.

[25] Ahn J.H., Choi Y.S., Choi J.H.: “Leptin promotes human endometriotic cell migration and invasion by up-regulating MMP-2 through the JAK2/STAT3 signaling pathway”. Mol. Hum. Reprod., 2015, 21, 792-802.

[26] Fidler I.J.: “Cancer metastasis”. Br. Med. Bull., 1991, 47, 157.

[27] Wang T., Xu H., Qi M., Yan S., Tian X.: “miRNA dysregulation and the risk of metastasis and invasion in papillary thyroid cancer: a systematic review and meta-analysis”. Oncotarget, 2017, 9, 5473-5479.

[28] Mei J., Wang D.H., Wang L.L., Chen Q., Pan L.L., Xia L.: “MicroRNA-200c suppressed cervical cancer cell metastasis and growth via targeting MAP4K4”. Eur. Rev. Med. Pharmacol. Sci., 2018, 22, 623-631.

[29] Harquail J., Benzina S., Robichaud G.A.: “MicroRNAs and breast cancer malignancy: an overview of miRNA-regulated cancer processes leading to metastasis”. Cancer Biomark., 2012, 11, 269-280.

[30] Li Y., Jiang Q., Xia N., Yang H., Hu C.: “Decreased expression of microRNA-375 in nonsmall cell lung cancer and its clinical significance”. J. Int. Med. Res., 2012, 40, 1662-1669.

[31] Wang F., Li Y., Zhou J., Xu J., Peng C., Ye F., et al.: “miR375 is down-regulated in squamous cervical cancer and inhibits cell migration and invasion via targeting transcription factor SP1”. Am. J. Pathol., 2011, 179, 2580-2588.

[32] Liu S.L., Sui Y.F., Lin M.Z.: “MiR375 is epigenetically downreg-ulated due to promoter methylation and modulates multidrug resistance in breast cancer cells via targeting YBX1”. Eur. Rev. Med. Pharmacol. Sci., 2016, 20, 32233229.

[33] Jung H.M., Patel R.S., Cheng J.Q., Chan E.K.L.: “Abstract 122: Downregulated miR375 in tongue cancer and its putative role as a tumor suppressor microRNA”. Cancer Res., 2011, 71, 122.

[34] Cui F., Wang S., Lao I., Zhou C., Kong H., Bayaxi N., et al.: “miR375 inhibits the invasion and metastasis of colorectal cancer via targeting SP1 and regulating EMT-associated genes”. Oncol. Rep., 2016, 36, 487-493.

[35] Li H., Lu Y., Pang Y., Cheng X., Chen J.: “Propofol enhances the cisplatin-induced apoptosis on cervical cancer cells via EGFR/JAK2/STAT3 pathway”. Biomed. Pharmacother., 2017, 86, 324- 333.

[36] Yang C., Zhang W., Wang L., Kazobinka G., Han X., Li B., et al.: “Musashi-2 promotes migration and invasion in bladder cancer via activation of the JAK2|[sol]|STAT3 pathway”. Lab. Invest., 2016, 96, 950-958.

[37] Kim B., Kim H.S., Kim S., Haegeman G., Tsang B.K., Dhanasekaran D.N., et al.: “Adipose stromal cells from visceral and subcutaneous fat facilitate migration of ovarian cancer cells via IL6/JAK2/STAT3 pathway”. Cancer Res. Treat., 2017, 49, 338-349.

[38] Colomiere M., Findlay J., Ackland L., Ahmed N.: “Epidermal growth factor-induced ovarian carcinoma cell migration is associated with JAK2/STAT3 signals and changes in the abundance and localization of alpha6beta1 integrin”. Int. J. Biochem. Cell. Biol., 2009, 41, 1034-1045.

[39] Vij N., Sharma A., Thakkar M., Sinha S., Mohan R.R.: “PDGF-driven proliferation, migration, and IL8 chemokine secretion in human corneal fibroblasts involve JAK2-STAT3 signaling pathway”. Mol. Vis., 2008, 14, 1020-1027.

[40] Zheng Q., Han L., Dong Y., Tian J., Huang W., Liu Z., et al.: “JAK2/STAT3 targeted therapy suppresses tumor invasion via disruption of the EGFRvIII/JAK2/STAT3 axis and associated focal adhesion in EGFRvIII-expressing glioblastoma”. Neuro. Oncol., 2014, 16, 1229-1243.

[41] Zhang X and Yin P., Luo G., Zheng L., Wei J., Zhang J., et al.: “IL6 regulates MMP-10 expression via JAK2/STAT3 signaling pathway in a human lung adenocarcinoma cell line”. Anticancer Res., 2009, 29, 4497-4501.

[42] Kim S., Choi J.H., Lim H.I., Lee S.K., Kim W.W., Cho S., et al.: “EGF-induced MMP-9 expression is mediated by the JAK3/ERK pathway, but not by the JAK3/STAT-3 pathway in a SKBR3 breast cancer cell line”. Cell Signal., 2009, 21, 892-898.

[43] Byun H.J., Darvin P., Kang D.Y., Sp N., Joung Y.H., Park J.H., et al.: “Silibinin downregulates MMP2 expression via Jak2/STAT3 pathway and inhibits the migration and invasive potential in MDA-MB-231 cells”. Oncol. Rep., 2017, 37, 3270-3278.

[44] Xu Y., Jin J., Liu Y., Huang Z., Deng Y., You T., et al.: “Snail-regulated MiR375 inhibits migration and invasion of gastric cancer cells by targeting JAK2”. PLoS One, 2014, 9, e99516.